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UNIVERSITY  OF  ILLINOIS 

Agricultural  Experiment  Station 


BULLETIN  NO.  161 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE 
ANIMAL  BODY 


BY  CHAS.  F.  BRISCOE 


URBANA,  ILLINOIS,  NOVEMBER,  1912 


LETTER  OF  TRANSMITTAL 

University  of  Illinois,  June,   1912. 
DR.  EUGENE  DAVENPORT, 

Director  of  Hie  Agricultural  Experiment  Station. 

Sir :  I  have  the  honor  to  transmit  and  recommend  for  publication  the 
accompanying  paper  prepared  under  my  direction  by  Chas.  F.  Briscoe,  Ph.D., 
upon  the  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY. 

This  is  a  companion  bulletin  with  No.  149  of  the  University  of  Illinois 
Agricultural  Experiment  Station  issued  under  date  of  February,  1911,  entitled 
TUBERCULOSIS  OF  FARM  ANIMALS, 'by  Chas.  F.  Briscoe  and  W.  J.  MacNeal. 
That  bulletin  was  based  upon  a  general  study  of  the  disease  in  bovine  ani- 
mals— its  characteristics,  modes  of  dissemination  and  infection,  methods  of 
recognition  and  prevention,  and  the  relation  to  tuberculosis  in  man. 

The  import  of  the  present  paper  is  well  expressed  in  the  title,  with  the 
understanding  that  the  studies  were  made  principally  upon  the  bovine  type  of 
the  causative  organism.  Much  previous  work  had  been  done  upon  the  same 
subject,  as  is  indicated  in  the  literature  herein  cited,  but  important  contribu- 
tions to  existent  knowledge  are  made  in  the  results  of  the  experiments  em- 
bodied in  this  publication,  while  earlier  announcements  have  been  confirmed 
or  disproved.  The  whole  matter  is  of  such  tremendous  significance  that  any 
additions  to  knowledge  and  any  further  dissemination  of  knowledge  upon  the 
subject  is  of  wide  and  vital  importance.  The  information  here  presented  will 
be  welcomed  by  specialists,  sanitarians,  and  the  general  public. 

T.   J.   BURRILL, 

Chief  in  Botany  {including  Bacteriology). 


SUMMARY  oi>  BULLETIN  No.  161 

1.  There  are  four  recognized  types  of  tubercle  bacilli;  human, 
bovine,  avian,  and  a  type  that  infects  cold-blooded  animals.     Only 
the  first  two  types  have  any  important  part  in  the  infection  of  man. 

Page  284 

2.  The  beaded  appearance  of  these  germs  led  the  early  inves- 
tigators to  a  belief  in  spore- formation.     This  is  now  known  not  to 
occur.     The   fatty  content  of   the  bacilli   varies    from    10  to   42 
percent,  which  is  five  times  as  much  as  found  in  any  other  micro- 
organism.    It  appears  that  this  fatty  material  has  little  or  nothing 
to  do  with  the  duration  of  their  viability.  Page  284 

3.  The  tubercle  bacillus  does  not  secrete  a  soluble  toxin,  but 
that  poisons  are  formed  is  well  known.     It  has  been  shown  by 
various  investigators  that  tubercles  can  be  produced  in  test  animals 
by  the  injection  of  dead  cultures.  Tubercles  thus  produced  may  be 
mistaken  for  those  produced  by  living  germs.  Page  284 

4.  A  clear  criterion  of  death  is  necessary  in  reporting  results 
on  the  duration  of  life  of  the  tubercle  bacillus.     This  fact  many 
investigators  have  disregarded.  Page  285 

5.  The    tubercle  bacillus  may  be    classed  with  the    nonspore- 
bearing  organisms  as  to  viability;   but  in  this  class  it  is  one  of  the 
most  resistant,  especially  as  to  drying  and  to  the  antagonism  of 
decay  organisms  in  water  ~and  foul  matter.  Page  286 

6.  The  action  of  sunlight  upon  bacteria  was  first  studied  by 
Downes  and  Blunt  in   1877.     Numerous  investigators  have  con- 
tinued this  study  to  the  present  day.  One  of  the  most  important 
facts  brought  out  is  that  bacteria  when  directly  exposed  to  the 
sun  are  killed  in  a  few  minutes.     This  is  due  to  the  effect  of  the 
ultra  violet  light.     The  ultra  violet  rays  are  now  cheaply  produced 
artificially  by  the  mercury  vapor  lamp.     This  lamp  is  destined  to 
play  an  important  part  in  sterilization  and  disinfection. 

Pages  287-291 

7.  It  is  shown  by  the  results  of  all  the  investigators  given  in 
Table  2  that  tubercle  bacilli  when  exposed  directly  to  the  sun  are 
killed  in  a  few  minutes  to  a  few  hours.     The  time  .of  killing  is 
less  at  higher  altitudes;    but  it  is  ten  to  fifteen  times  longer  in 
diffuse  light.  Pages  292-295 

8.  Tuberculous  sputum   reduced  to  dust  and  inhaled  by  test 
animals  causes  tuberculosis.     A  much  less  amount  is  necessary  to 
produce  tuberculosis  by  inhalation  than  by  ingestion.     This,  how- 

279 


ever,  must  not  be  taken  to  indicate  that  inhalation  plays  a  more 
important  part  than  ingestion  as  a  cause  of  tuberculosis.  The 
more  important  method  depends  upon  the  opportunity  of  infection 
from  each.  Investigators  do  not  agree  on  this  question.  At  pres- 
ent both  inhalation  and  ingestion  should  be  considered  dangerous 
sources  of  tuberculous  infection.  Pages  295-306 

9.  The   Mills-Reincke   phenomenon,   which   has  been   given   a 
mathematical   equivalent  by   Hazen's   theorem,   viz.,   "Where  one 
death  from  typhoid  fever  has  been  avoided  by  the  use  of  better 
water,  a  certain  number  of  deaths,  probably  two  or  three,  from 
other  causes  have  been  avoided,"  has  been  found  by  Sedgwick  and 
MacNutt  to  be  sound  and  conservative.    Their  studies  show  that 
tuberculosis  has  decreased  in  certain  cities  of  Massachusetts,  which 
decrease  is  evidently  due,  in  part,  to  the  improvement  of  their 
water  supply.  Pages  307-308 

10.  It  is  reported  in  the  literature  that  tubercle  bacilli  live  for 
a  very  long  time,  several  months  to  more  than  a  year,  in  water  and 
other  material.  Pages  309-310 

11.  In  experiments  to  determine  the  time  that  tubercle  bacilli 
live    in    various  conditions  the  chief  difficulty  is    the  "index  of 
death"    for  these  germs.   This  is    true  since    cultivation  of    the 
tubercle  bacilli   from  contaminated  material   is  not   feasible,   and 
since  the  dead  germs  produce,  in  test  animals,  tubercles  indistin- 
guishable by  microscopic  appearance  from  those  produced  by  live 
tubercle  bacilli.  Pages  311-313 

12.  Pure  cultures  of  nonspore-bearing  organisms  and  the  vege- 
tative cells  of  spore-bearing  germs  when  exposed  to  direct  sun- 
light in  thin    smears  are  killed  in    l/>  to  6  minutes ;    the  human, 
bovine,  and  avian  types  of  tubercle  bacilli  exposed  in  the  same  way 
were  killed  in  i  to  4  minutes.  Pages  314-317 

13.  When   exposed   to   desiccation    in   a   dark,   well-ventilated 
place,  the  nonspore-bearing  organisms  and  the  vegetative  cells  of 
spore-bearing  organisms  died  in  i  to  4  days;    spores  of  B.  subtilis 
and  B.  vulgatus  used  as  controls  were  not  killed  in  35  days ;    the 
human  and  bovine  type  of  tubercle  bacilli  exposed  at  the  same  time 
and  under  the  same  conditions  were  dead  within  4  and  8  days  re- 
spectively. Pages  317-318 

14.  Pure  cultures    of  bovine    tubercle  bacilli    mixed  in    cow 
manure  and  exposed  in  a  two-inch  layer  in  a  pasture  field  in  the 
sunshine  remained  alive  and  virulent  for  two  months. 

Pages  323-324 

280 


15.  As  would  be  expected,  these  germs  exposed  in  cow  manure 
retained  their  virulence  longer  in  the  shade  than  in  the  sunshine, 
as  shown  both  by  the  greater  severity  of  the  disease  produced  in 
the  guinea  pigs  inoculated  with  the  germs  exposed  in  the  shade, 
than  that  produced  in  the  guinea  pigs  inoculated  on  the  same  day 
with  the  germs  exposed  in  the  sunshine,  and  by  the  greater  length 
of  time  that  the  guinea  pigs  which  were  inoculated  with  the  germs 
exposed  in  the  sunshine  remained  alive.  Pages  325-326 

1 6.  Tubercle  bacilli  in  the  manure  of  a  naturally  infected  cow 
exposed    in  the  same  manner  as  the    artificially  infected    manure 
were  dead  within  two  weeks  after  exposure.  Pages  327-334 

17.  Tubercle  bacilli  in  garden  soil  and  in  a  dead  tuberculous 
guinea  pig  buried  in  garden  soil  were  alive  on  the  21 3th  and  the 
7  ist  days,   respectively,  and  dead  on  the  23Oth  and    99th    days, 
after  first  exposed.  Pages  334-339 

1 8.  Tubercle  bacilli  live  for  more  than  a  year  in  running  water. 
A  watering  trough  harboring  these  germs  may  be  a  dangerous 
source  of  infection  to  cattle.     The  better  disposition  of  dead  tuber- 
culous animals  is  to  destroy  by  burning.     Tubercle  bacilli  in  drink- 
ing water  is  one  of  the  possible  sources  of  infection  for  man.     In- 
fection  is  not  prevented  by  dilution,   since  clumps   containing  a 
great  number  of  these  organisms  may  be  inclosed  in  mucoid  ma- 
terial which  prevents  their  separation  and  destruction. 

Pages  340-359 

19.  Tubercle  bacilli  in  market  butter  placed  in  cold  storage  live 
for  more  than  ten  months,  which  is  a  longer  time  than  such  butter 
is  usually  kept  in  storage.  Pages  359-363 

20.  General  discussion.  Pages  364-365 

21.  References.  Page  366 


281 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE 
ANIMAL  BODY 

BY  CHAS.  F.  BRISCOB,  INSTRUCTOR  IN  BOTANY1 

INTRODUCTION 

The  fact  that  one-tenth  of  all  deaths  in  the  human  family  are 
due  to  tuberculosis,  and  that  millions  of  dollars  worth  of  farm 
animals  are  lost  annually  from  its  ravages,  makes  any  knowledge 
concerning  the  fate  of  tubercle  bacilli  outside  of  the  animal  body 
of  great  value. 

These  questions  have  often  come  from  stock  owners  :  How 
long  is  it  necessary  to  keep  healthy  stock  from  a  field  where  tuber- 
culous cattle  have  been  previously  allowed  to  run?  How  long  do 
tubercle  bacilli  live  in  manure,  in  a  watering  trough,  and  in  a  dead 
tuberculous  animal  ?  And  again,  some  authors  have  questioned  the 
reports  in  the  literature  that  tubercle  bacilli  remain  alive  and  viru- 
lent for  periods  of  a  year  and  more  outside  of  the  animal  body, 
They  have  suspected  that  in  such  cases  the  tubercles,  found  in  the 
test  animals  after  inoculation  with  such  tuberculous  material,  kept 
for  these  long  periods,  had  been  produced  by  dead  tubercle  bacilli. 
It  has  been,  in  part,  the  purpose  of  this  bulletin  to  answer  these 
questions. 

The  first  section  is  devoted  to  brief  notes  on  the  biology  of  the 
tubercle  bacillus;  then  a  tabular  review  of  the  literature  is  given 
\vith  brief  discussions;  and  lastly  an  account  of  the  experimental 
work,  followed  by  a  general  discussion.  The  experiments  deal 
with  pure  cultures  of  tubercle  bacilli,  and  with  those  of  some  other 
bacteria  for  purposes  of  comparison  as  to  their  duration  of  life  in 
sunlight  and  under  desiccation;  and  with  the  time  -limit  of  life  of 
tubercle  bacilli  in  cow  manure,  garden  soil,  water,  butter,  and  dead 
tuberculous  animals. 

BIOLOGY  OF  THE  TUBERCLE  BACILLUS 


MORPHOI  OGY  tubercle  bacillus  varies  in  form  according  to 

type,  method  of  growth,  and  age  of  the  individual 
culture.  It  is  a  slightly  curved,  rod-shaped  organism  measuring 
from  0.3  to  0.5  microns  in  diameter  and  from  2  to  5  microns  in 

'Now   Professor  of  Bacteriology  in   the  Mississippi   Agricultural   Experiment  Station, 
Agricultural  College,  Mississippi. 

283 


284  BULLETIN  No.  161  [November. 

length.  There  are  two  well-recognized  types  that  infect  mammals, 
the  human  and  the  bovine.  The  latter  is  shorter  and  thicker  and 
stains  more  uniformly.  The  avian  type,  found  in  domestic  fowls 
and  birds,  and  a  type  that  infects  the  cold-blooded  animals,  espe- 
cially fishes  and  frogs,  play  almost  no  part  in  the  infection  of  man. 
The  tubercle  bacillus  often  presents  a  beaded  appearance.  This 
is  more  common  in  the  specimens  found  in  old  pus  and  sputum.  It 
is  due  to  a  fragmentation  of  the  protoplasm.  This  peculiar  struc- 
ture of  the  organism  led  the  earlier  scientists,  to  the  belief  in  spore 
formation.  Koch74,  in  his  first  paper,  Die  Aetiology  der  Tubercn- 
los,  held  to  this  belief,  and  this  idea  of  spore  formation  has  found 
place  in  many  publications  even  to  the  present  day.  It  is  now  known, 
from  the  relation  of  these  organisms  to  the  action  of  heat,  sun- 
shine and  chemical  disinfectants,  that  they  do  not  form  spores 
They  are  killed  very  readily,  in  thin  layers,  by  the  direct  rays  of 
the  sun,  and  also  at  a  temperature  of  60°  C.  in  fifteen  to  twenty 
minutes.  In  these  respects  they  are  like  other  nonspore-bearing 
organisms. 

Their  power    of  resistance  to    drying  and  to    the 

WAXY  OR  FATTY  v.  ,  .         J 

SUBSTANCE  antagonism  of  decay  organisms  appears  to  be 
greater  than  that  of  other  nonspore-bearing  or- 
ganisms, tho  less  than  that  of  the  spore1  organisms.  This  power 
of  resistance  is  no  doubt  due,  in  part  at  least,  to  the  content  of 
waxy  or  fatty  substance  found  largely  in  the  outer  layer  of  the 
tubercle  bacillus.  The  presence  of  this  waxy  material  gives  them 
their  well-known  character  of  "acid  prqof"  power  when  stained. 
(When  this  waxy  or  fatty  substance  is  extracted  with  ether,  stains 
are  no  longer  held  on  treatment  with  an  acid  or  alcohol.)  The 
bacillus  has  the  largest  amount  of  fatty  substance  of  any  known 
micro-organism.  The  fat  content  varies,  according  to  different  in- 
vestigators, from  10  to  42  percent;  while  in  other  micro-organisms 
an  alcohol  ether-extractive  has  been  found  to  vary  only  from  1.7  to 
i  o.i  percent. 

The  tubercle  bacillus  does  not  secrete  a  soluble 
THE3(TNuSBERCLE  toxin>  as  do  B-  diphtheria  and  B.  tetam.  It  has  not 
BACILLUS  "  been  demonstrated  that  the  tubercle  bacillus  forms 

a  true  toxin.  Levene87  proved  the  absence  of  tox- 
albumins  from  extracts  of  the  bacillus.  That  poisons  are  formed 
is  well  known,  tho  their  character  is  not  understood.  Baldwin10 
thinks  that  the  symptoms  and  toxemia  of  tuberculosis  are  accounted 
for  by  the  presence  of  the  nucleic  acid  products  in  the  blood. 
Koch73,  Von  Prudden  and  Hodenpyle155,  Vissman154,  Straus  and 
Gamaleia144,  Sternberg145,  Krompecher75,  Miller97,  and  Rosenau121 


I91-]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  283 

have  produced  tuberculosis  in  test  animals  (guinea  pigs  and  rab- 
bits) by  injecting  dead  cultures.  These  results  have  been  confirmed 
in  the  laboratory  here.  (See  experiments  page  312). 

The  injection  of  dead  cultures  killed  by  heat  or 
DEA'D  CULTURE s^  chemical  disinfectants  will  produce  necrosis, 

abscesses,  caseation,  emaciation,  and  death.  So 
closely  do  these  lesions  resemble  those  produced  by  the  living  germs 
that  it  is  difficult  to  know  whether  we  are  dealing  with  dead  or  with 
living  cultures.  JRosenau123  suggests  that  much  of  the  work  done 
upon  the  duration  of  life  of  tubercle  bacilli  has  little  value  for 
lack  of  a  clear  criterion  of  death.  Lesions  from  dead  cultures,  as 
from  live  ones,  may  be  characterized  by  giant  cells.  Tuberculin 
cannot  be  depended  upon  to  distinguish  between  the  lesions  pro- 
duced by  dead  tubercle  bacilli  and  those  produced  by  live  germs. 
The  reactions  caused  by  the  injection  of  2  cc.  of  tuberculin  under 
the  skin  of  guinea  pigs  with  lesions  produced  by  dead  cultures  are 
similar  to  those  caused  by  a  like  injection  into  guinea  pigs  with 
lesions  produced,  from  living  cultures,  even  to  the  death  of  the 
guinea  pig. 

Most  investigators  have  not  taken  into  account  the  fact  that 
dead  tubercle  bacilli  produce  lesions,  and  in  reporting  their  results 
have  taken  for  granted,  when  at  autopsy  even  local  lesions  have 
been  found,  that  the  tubercle  bacilli  have  been  alive  and  virulent. 
The  only  safe  method  of  distinguishing  is  by  inoculating  a  second- 
ary healthy  guinea  pig  with  a  small  amount  of  the  tuberculous 
material  from  the  lesions  of  the  original  test  animal.  If  the  bac- 
teria are  alive,  there  develops  a  generalized  tuberculosis,  usually 
severe;  if  dead,  either  no  lesions  at  all  or  only  a  slight  localized 
effect  will  occur.  Cultivations  from  this  tuberculous  material  from 
both  the  original  and  the  secondary  test  animal  will  give  further 
evidence.  If  the  organisms  are  dead,  no  growth  occurs;  if  alive, 
growth  is  usually  evident.  This  method,  tho  time  consuming,  is 
"ecessary  to  obtain  the  most  trustworthy  information. 

That  the  tubercle  bacillus  does  not  possess  the  re- 
R  EL  ATI  ON  OF  si  stance  to  external  agents  as  do  the  spore-bearing 
QILLI  TO  OTHER  organisms  is  unquestioned.  The  spores  of  Bact. 
ORGANISMS  anthracis  have  been  known  to  hold  their  virulence 

from  ten  to  twelve  years  (Aiello  and  Drago2). 
We  have  found  that  the  spores  of  B.  subtilis  dried  on  an  agar  slant 
and  remaining  in  this  state  for  eight  years,  gave  growth  when 
seeded  into  broth.  It  might  be  expected,  however,  that  tubercle 
bacilli,  from  their  waxy-fatty  content  and  from  their  analogy  to 
spores  in  respect  to  staining  qualities,  would  be  more  resistant  to 


286  BULLETIN  No.  161  [\rorember, 

external  injurious  agencies  such  as  heat,  drying,  light,  chemicals, 
and  putrefaction.  This,  in  general,  is  found  not  to  be  the  case. 
Rosenau123  says,  "The  tul>ercle  bacillus  may  be  classed  with  the 
nonspore-bearing  organisms  so  far  a"s  its  viability  is  concerned." 
This  statement  is  surely  correct  with  respect  to  heat  and  light. 
The  thermal  death  point  of  the  tubercle  bacillus,  as  determined  by 
the  more  careful  investigators  (Herr50,  1901 ;  Hess57,  1901 ;  Rus- 
sell and  Hastings  127,  1904;  and  Rosenau  121,  1908),  is  given  at 
60°  C.,  with  an  exposure  of  from  fifteen  to  twenty  minutes.  This 
is  practically  the  same  as  the  thermal  death  point  determined  for 
most  other  nonspore-bearing  organisms.  (Sternberg145,  Smith130). 

As  regards  desiccation,  and  more  especially  the  antagonism 
offered  by  decay  and  foul  matter,  tubercle  bacilli  appear  rather  to 
take  an  intermediate  position  between  spore-  and  nonspore-bearers. 
Rickards,  Slack  and  Arms119  found  that  tubercle  bacilli  in  sputum 
resisted  drying  for  88  days;  LeNoir  and  Camus83  found  these 
organisms  alive  after  33  days;  Kuss77  found  them  alive  from  20 
to  30  days,  and  killed  in  40  to  60  days;  Noetel105  found  them  yet 
living  after  35  days.  Maffucci90  shows  that  pure  cultures  of  avian 
tubercle  bacilli  dried  on  silk  threads  live  for  14  days.  Kirstein69 
finds  pure  cultures  dried  in  dust  living  from  3  to  8  days.  In  my 
own  experiments  they  lived  8  days.  Other  nonspore-bearing  organ- 
isms die  in  a  very  short  time,  as  B.  ziolaceus,  I  day ;  B.  "typhoid, 
3  days;  and  B.  coli,  3  days.  There  are  reported  in  the  litera- 
ture cases  where  nonspore-bearing  organisms  have  resisted  drying, 
under  special  conditions,  for  a  very  long  time.  Rosenau123  finds 
B.  pestis  to  live  over  four  months  on  a  piece  of  dry  sponge. 
Sirena  and  Alessi135  report  that  the  pneumonia  diplococcus,  when 
dried  on  silk  threads  kept  in  a  moist  room,  did  not  die  until  after 
192  days.  More  comparative  work  must  be  done  with  cultures  of 
different  nonspore-bearers  and  with  tubercle  bacilli  exposed  under 
the  same  conditions  before  definite  conclusions  can  be  drawn. 

In  the  presence  of  foul  material  tubercle  bacilli  live  from  a 
month  to  a  year  and  more  (see  Table  7).  In  my  own  experi- 
ments they  were  always  found  to  live  in  water  for  202  days  and 
as  long  as  441  days.  In  this  work  much  care  was  taken  to  deter- 
mine that  the  tuberculosis  produced  in  the  -test  animals  was  by 
living  germs.  Careful  workers  like  Jordan,  Russell  and  Zeit65  have 
found  B.  typhosus  living  in  sewage  from  3  to  4  days  only;  Russell 
and  Fuller126  report  from  8  to  TO  days.  It  .would  be  expected 
that  tubercle  bacilli  protected  by  the  mucoid  material,  as  found  in 
sputum  and  diseased  tissues,  in  which  these  germs  more  frequently 
occur,  and  also  by  their  abundance  of  naturally  waxy  constituents, 


191-}  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  AXIMAL  BODY  287 

would  be  protected  against  drying  and  injuries  from  the  presence 
of  foul  material.  This  conclusion  is  well  borne  out  by  the  litera- 
ture and  by  the  experimental  data  given  in  this  bulletin. 

REVIEW  AND  DISCUSSION  OF  THE  LITERATURE 

THE  ACTION  OF  SUNLIGHT  ON  BACTERIA 

Historical 

The  killing  power  of  sunlight  on  bacteria  was  first  noted  by 
Downes  and  Blunt34  in  1877.  They  worked  with  mixed  cultures 
suspended  in  culture  solutions  and  found  that  the  organisms  were 
killed  in  a  short  time,  as  shown  by  the  solutions  remaining  clear. 
Tyndall150  questioned  the  results  of  Downes  and  Blunt  but  later 
published  results151  confirming  them.  Duclaux37  (1879)  was  the 
first  to  test  the  action  of  sunlight  upon  pure  cultures  and  thus  place 
the  work  upon  a  scientific  basis.  Arloing7  (1885)  was  the  first  to 
test  the  action  of  light  upon  pathogenic  bacteria  (Bact.  anthracis}. 
He  was  also  the  first  to  use  the  electric  light  in  sujcji  experimental 
work.  Feltz38  (1890)  was  the  first  to  test  the  action  of  light  upon 
tubercle  bacilli.  The  classic  works  of  Buchner16  and  Ward157 
(1892-3)  deserve  special  mention.  Buchner  was  the  first  to  ex- 
hibit the  killing  power  of  light  in  reproducing  characters  by  the 
growth  of  the  organisms  in  that  part  of  the  Petri  dish  protected 
from  the  sunlight.  The  organisms  are  thickly  seeded  in  a  solid 
medium  like  agar,  and  the  desired  characters  cut  from  black  paper 
are  fastened  over  this  Petri  dish.  The  organisms  exposed  to  the 
sunlight  are  killed ;  where  protected  by  the  black  paper,  they  grow, 
forming  the  letters.  Ward  called  especial  attention  to  the  effect 
of  different  rays  of  the  sun,  which  he  separated  by  the  use  of 
screens  of  colored  glass  and  colored  solutions. 

There  early  arose  the  questions :  To  what  is  the  killing  of  bac- 
teria due?  and,  Are  spores  more  easily  killed  than  the  vegetative 
cells?  Arloing7  (1885)  showed  that  spores  of  Bact.  anthracis  in 
broth  were  killed  in  two  hours,  while  to  kill  the  vegetative  cells 
required  from  twenty-six  to  thirty  hours.  Nocard104  (1885)  sug- 
gested that  during  the  exposure  the  spores  developed  into  bacilli 
and  the  light  acted  upon  the  more  sensitive  vegetative  cells. 
Straus141  (1886) 'apparently  confirmed  this  suggestion  of  Nocard 
by  exposing  anthrax  spores  in  broth  and  in  distilled  water.  He 
found  that  the  spores  in  the  broth  were  killed  in  nine  hours  while 
those  in  distilled  water  were  not  killed  in  this  time.  In  1886  Ar- 
loing* repeated  his  experiments,  exposing  the  cultures  on  ice  so 
that  the  spores  could  not  develop,  and  confirmed  his  earlier  results. 
Roux125  (1887)  finds  the  explanation  in  the  fact  that  spores  are 
more  readily  killed  by  the  action  of  sunlight  in  the  presence  of 


288  BULLETIN  No.  161  [November. 

oxygen  than  when  the  oxygen  is  excluded.  He  thinks  that  from 
the  nature  of  the  spore  constituents  more  oxidation  products  are 
formed  in  the  spore  chan  in  the  vegetative  cell,  and  that  for  this 
reason  the  spore  is  more  readily  killed.  Dieudonne31  (1894)  con- 
cludes that  the  formation  of  hydrogen  peroxid  by  the  action  of 
sunlight  on  the  presence  of  oxygen  forms  an  important  part  in  the 
killing  of  bacteria.  Kruse76  (1895)  and  Richardson118  (1893) 
also  came  to  this  same  conclusion.  Thiele  and  Wolf147  ( 1906) 
carried  out  carefully  planned  experiments  in  which  their  cultures 
of  B.  prodigiosus,  B.  pyocyaneus  and  B.  coli  were  exposed  in  the 
presence  of  air,  oxygen,  or  hydrogen.  They  took  special  precau- 
tions to  confine  the  gases  used,  with  mercury  joints,  so  as  not  to 
allow,  the  slightest  diffusion.  They  found  that  the  bacteria  were 
killed  as  readily  anaerobically  as  aerobically,  and  conclude,  there- 
fore, that  the  killing  of  the  bacteria  by  light  is  not  assignable  to 
the  indirect  influence,  of  oxidation  of  water.  They  exposed  their 
cultures  in  broth  diluted  i-ioqo,  physiological  salt  solution  and 
Elbe  river  water. 

It  can  readily  be  shown  that  by  the  exposure  of  physiological 
salt  solution  to  sunlight  for  the  length  of  time  the  investigators  ex- 
posed their  cultures  there,  is  formed  some  condition  sufficient  to 
kill  bacteria.  The  question  whether  the  bactericidal  action  is  due 
to  an  injurious  chemical  formed  by  the  action  of  the  sunlight,  or 
whether  it  is  a  direct  bactericidal  action  of  the  light,  is  still  unset- 
tled. The  facts  thus  far  established  indicate  that  the  action  is  due 
to  both  a  chemical  and  a  direct  bactericidal  action,  sometimes  one 
and  sometimes  the  other  being  predominant,  depending  upon  the 
condition  of  the  experiment.  A  more  important  fact,  which  has 
been  especially  emphasized  in  a  recent  publication  (see  Weinzirl, 
Table  2)  is  that  bacteria  when  directly  exposed  to  sunlight  are 
killed  in  a  few  minutes,  in  contrast  to  most  of  the  published  results 
of  a  few  hours  to  a  few  days.  This  is  due  to  the  well-known 
germicidal  effect  of  the  ultra  violet  rays.  Since  glass  is  an  excel- 
lent screen  for  these  rays,  an  exposure  of  bacteria  in  glass  contain- 
ers lengthens  very  greatly  the  time  necessary  to  kill. 

Powerful  beams  of  ultra  violet  rays  are  now  artificially  pro- 
duced with  the  quartz  mercurv  vapor  lamp.  A  number  of  these 
machines  are  upon  the  market.  The  rays  from  them  are  so  effec- 
tive that  bacteria  directly  exposed  in  thin  layers  are  killed  almost 
instantly.  Indeed,  so  strong  are  the  most  powerful  of  these  lamps 
that  the  skin  accidently  exposed  is  killed  and  later  sloughs  off. 

It  is  stated  that  the  sterilization  of  a  city  water  supply  can  be 
done  economically  with  these  lamps.  A  test5  at  Marseilles,  France, 
shows  that  the  cost  per  million  gallons  of  water  is  only  ten  dollars. 


1913] 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


289 


i.  e.,  one  cent  per  1000  gallons.  This  indicates  a  feasible  system  of 
supplying  a  city  with  a  good,  potable  water  from  a  contaminated 
source.  Years  ago  it  was  suggested  by  Marshall  Ward  that  the  dis- 
infection of  cow  sheds  might  well  be  done  with  electric  lights. 
Tho  this  was  thought  fanatical  at  the  time,  it  may  be  feasible  with 
such  light  as  the  present  powerful  mercury  vapor  lamp  produces. 
Why  cannot  the  "toning  up"  effect  that  is  to  be  felt  in  a  room 
which  has  been  exposed  to  the  sunlight  thruout  the  day  be  in  a 
way  brought  about  by  exposing  to  one  of  these  powerful  mercury 
vapor  lamps,  for  a  few  hours  in  the  evening,  such  rooms  as  have 
no  access  to  the  sun,  at  the  same  time  securing  the  great  benefit 
that  comes  from  disinfection  by  its  powerful  beams? 

For  further  discussion  of  this  most  interesting  subject  we  refer 
our  readers  to  the  recent  numbers  of  the  periodicals,  more  espe- 
cially the  chemical  and  engineering  journals.  The  following  table 
gives  a  brief  review  of  the  results  found  in  the  literature  upon  the 
action  of  sunlight  on  bacteria. 


TABLE  1 — ACTION  OF  SUNLIGHT  UPON  BACTERIA 


Author 

Date 

Conditions 

Not 
killed 

Killed 

Dowries 

1877 

Cultures  in  Pasteur  solutions  

1879 

Tyrothrix  filiformis  spores    

Duclaux36 

1885 

Tyrothrix  scaber  from  milk: 
A  drop  dried  in  an  empty  flask  
In  broth  culture  vegetative  cells.  .  . 
Kept  in  the  dark  

14  days 
3  yrs 

60  days 
15  days 

Duclaux36 

1885 

•  Micrococci,  young  cultures  veal  broth: 
In  dark  

40  days 

In  sunshine,  July  .....    

jn  dried  condition  

8.  3  and  even  2  days 

Arloing6 

18S5 

Bact.  anthracis  spores  in  broth  
Bact.    anthracis   vegetative    cells    in 
broth   

1  to  \Yt  hrs. 

2  hrs. 

after  2  hrs. 
26  to  30  hrs 

Straus141 

1886 

Bact.  anthracis  spores: 

9  hrs 

9  hrs. 

Arloing8 

18S6 

Bact.  anthracis  spores  in  broth  on  ice 
4°  c     

5  hrs 

Dowries" 

1886 

Bacteria  in    mixed    cultures,    diffuse 
litrht 

5  days 

Roux133 

1887 

Bact.  anthracis  spores: 
\Vithout  air  

83  hrs. 

With  air   . 

29  to  54  hrs. 

290 


BULLETIN  N.o.  161 
TABLE  1 — Continued 


[November, 


Author 

Date 

Conditions 

Not  killed 

Killed 

Pansini108 

1889 

B.  violaceus,  B.  prodigiosus,  B.  pyo. 
cyaneus,     Bact.      anthracis,     Msp 
comma,  Murisepticus,    and   Staph' 
pyogenes  aureus: 
Exposed  to  diffuse  daylight,  devel- 
opment hindered  

24to48hrs. 

Exposed  to  sunshine  in  tube  cultures 
In  hanging  drop  on  needle  

1  day. 
y*  to2j^  hrs 

L/aurent80 

1890 

Bacillus  of  Kiel  on  potato  

1  to  3  hrs. 

5  hrs 

Janowsky'8' 

1890 

B.    typhosus  in  culture  media  

4  to  10  hrs 

Buchner18 
Buchner15 

1892 
1893 

B.    typhosus  on  agar  plates      
B.    typhosus,    B.    pyocananeus,     and 
Msp.  comma  on  plates  1.6  meters 
under  water                      •           

1  to  \l/2  hrs. 
\y2  hrs 

Geisler80 

1892 

B.    typhosus  (almost  all  killed)  

3  to  6  hrs 

Moment100 

1892 

"  Asporegeiie  anthrax"  in  phenol  broth: 
Dried  and  exposed  in  air     

5  to  5j4  hrs 

Dried  and  exposed  in  vacuo  

6%  hrs. 

In  moist  condition  in  the  air  

2^  hrs. 

In  moist  condition    no  air       

50  hrs 

Dried  anthrax  spores  in  the  air  

100  hrs. 

Dried  anthrax  spores  in  vacuo  

100  hrs. 

Moist  anthrax  spores  in  the  air  .      ... 

44  hrs. 

Ward167 
Frankland  and 
Appleyard41 

1893 
1893 

Moist  anthrax  spores  in  vacuo  
Bact.  anthracis  spores  on  agar  
Bact.     anthracis    spores    in    Thames 
river  water: 
In  diffuse  daylight  1  

6  mo. 

100  hrs. 
4  to  10  hrs. 

In  direct  sunshine   

151  hrs. 

Bact.  anthracis  vegetative  cells: 
In  unsterilized  water     

56  hrs. 

84  hrs. 

L/edoux- 

In  sterilized  water  

84  hrs. 

L/ebard81 
Ward167 

1893 
1893 

B.  diphtheria  in  broth  culture    ... 
Organisms  on  agarinPetri  dishes  with 
quartz  glass  covers    

few  days 
2  to  6  hrs. 

Ward  and 
Cartwright158 

1893 

Bact.  anthracis  in  unsterilized  Thames 

d'Arsonval 

1894 

B    pyocyaneus  on  agar      

and  Charrin9 
Gaillard45 

1894 

3  to  4  hrs 

Dieudonne81 

1894 

B.  coli,  B.  typhosus,  Bact.  anthracis, 
B.  prodigiosus,  and  B.  fluorescens 
By   electric    arc    light    900    candle- 
power  

%  to  2%  hrs. 
8  hrs 

Bact.  anthracis  spores: 
In  the  air  

3^  hrs 

Air  excluded  ......               

3^  hrs 

B.  tetani  spores,  air  excluded  

9  hrs 

Kruse76    - 

1895 

Bact.    anthracis    spores    in    hanging 
drop  

\y2  hrs 

2  to  5  hrs 

B.  typhosus: 
In  broth   air  present   

iy2  to  7  hrs 

In  broth,  air  replaced  with  hydrogen 
Bact.  anthracis   diffuse  light     .  .  . 

7  hrs. 
(13  da3Ts 

SO  days) 

1  Tho  the  spores  were  not  killed,  they  were  so  weakened  that  one  cubic  centi- 
meter did  not  kill  mice.  A  broth  culture  of  the  same  in  much  smaller  quantity 
killed  mice.  ^ 


19 J-]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 

TABLE  1.—  Continued. 


291 


Author 

Date 

Conditions 

Not  killed 

Killed 

Beck  and 
Schultz1  a 

1896 

Bacteria    

short  time 

Gehrke49 

1899 

Bacteria:1 
In  clear  water    

2  to  8  hrs 

Agar  cultures  

6  hrs. 

Kedzior67 
Jones6  * 

1899 
1900 

B.    pyocyaneus,  B.    diphtheria,    Msp. 
metchnikovy,  and  spores  of  Bact. 
anthracis,  exposed  on  gelatin  .... 
B.  cartovorus  directly  exposed   

5  min. 

IX  to  3X  hrs. 
10  min. 

Mettler95 

1904 

Msp.  comma,  B.  typhosus,  and  B.  coli 
in  broth  culture  

few  hours 

Huber61 
Thiele147 
and  Wolf 

1905 
1906 

Streptococci  and  B.  diphtheria  in  broth 

B.   coli,    and   B.    typhosus   in  diluted 
broth,  Elbe  river  water,  or  physi- 
ological salt  solution     

2  to  3  hrs. 
9  hrs 

Weinzirl160 

1906 

.  Exposed   directly  to  the  sun  in  thin 
smears: 
B.  cholera  suis,  B.  prodigiosus,  B.  ty- 
phosus   

5  to  10  min 

B.  typhosus  (repeated),  B.  dysenteria, 
B.     prodigiosus      (repeated),      B. 

2  to  5  mill 

Msp   comma  

2  to  6  min 

B.  diphtheria  

5  min. 

Pus  cocci     -  ••' 

5  to  15  min 

B    coli       ...            

/*2  to  2  min 

B.  f  riedlJlnder        >•  • 

0  to  1  min. 

B.  phosphorescens      

1  to  2  min. 

Sar.  aurantiaca  

90  min. 

Pink  air  micrococci  

120  min. 

Neumark103 

1907 

Exposed   to    direct  sunlight    on  agar 
plate: 
B   anthracis  spores  

25  to  30  min 

B    anthracis  vegetative  cells  

20  to  25  min 

B    coli        

60  min. 

'  'Schweinpest  bacilli'  '  

110  to  120  min. 

Chicken  cholera  ......    

8  to  10  min. 

240  min. 

Orsit06 

1907 

B.  typhosus  and  Msp.  comma  in  sun- 
light 

8  to  10  hrs 

B    coli  in  distilled  water  

6  hrs. 

12  hrs. 

McNaughtarid 
Korich34 

1910 

B.  coli: 
In  distilled  water  in  air  at  0°  to  15°C. 
in  diffuse  daylight   

25  days 

In  strong  sunlight,  numbers  much 
decreased            

6  hrs. 

In  strong  sunlight  ...    

12  hrs. 

Dried  in  a  desiccator   . 

11  davs 

292  BULLETIN  No.  161  [November. 

THE  DURATION  OF  LIFE  OF  TUBERCLE  BACILLI  IN  SUNSHINE  AND 
IN  DIFFUSE  DAYLIGHT 

INDEX  OF  Every  author  consulted,  except  Weinzirl.  used  the 

DEATH  animal  test  to  determine  when  tubercle  bacilli  were 

dead.  \Veinzirl,  using  only  pure  cultures,  em- 
ployed the  method  of  cultivation.  No  investigator  whose  work  is 
recorded  in  Tables  2  and  3  has  called  attention  to  the  fact  that  dead 
cultures  produce  tuberculosis  (for  discussion  see  page  112).  Just 
what  error  a  neglect  of  attention  to  this  point  has  introduced  into 
the  results  given  in  the  following  table  cannot  be  definitely  known 
Tho  it  is  certain  that  dead  tubercle  bacilli  do  produce  tuberculosis, 
yet  in  no  case  was  it  found  in  my  own  experimental  work  that 
localized  tuberculosis  was  produced  by  dead  organisms.  In  every 
case  the  secondary  guinea  pigs  inoculated  from  cases  of  localized 
tuberculosis  became  tuberculous  and  usually  severely  infected.  Cul- 
tures from  diseased  tissues  produced  growth,  either  from  the  origi- 
nal or  from  the  secondary  inoculated  guinea  pigs,  or  from  both.  It 
appears,  therefore,  that  the  error  arising  from  not  taking  into 
account  tuberculosis  produced  by  dead  cultures  is  probably  slight. 

TIME  REQUIRED  Pure  cultures  of  tubercle  bacilli  when  exposed  in 
TO  KILL  thin  layers  to  the  direct  sunlight  are  killed  in  from 

a  few  minutes  to  a  few  hours.  This  has  been  the 
result  obtained  by  all  investigators.  To  kill  these  germs  in  sputum 
requires  only  a  slightly  longer  time.  The  mucoid  mass  is  a  slight 
protection  which  increases  with  the  thickness  and  opacity  of  the 
material,  but  even  here  tubercle  bacilli  are  killed  in  a  very  short 
time.  With  two  exceptions,  that  of  Feltz38  and  of  Mitchell  and 
Crouch98  (see  Table  2),  no  investigator  reports  these  bacilli  surely 
living  after  twenty-four  hours'  exposure  to  the  sun.  In  these  two 
cases  the  sputum  was  exposed  on  soil,  and  a  small  layer  of  soil 
would  afford  good  protection  from  the  action  of  the  sunlight. 

EFFECTOR  Experiments  definitely  planned  to  test  the  killing 

HIGH  ALTITUDE  power  of  the  sun  on  tubercle  bacilli  at  different 
altitudes  have  been  carried  out  by  a  Russian,  Tres- 
kinskaja148.  In  our  country  Mitchell  and  Crouch98  have  determined 
the  effect  of  the  sun  on  tuberculous  sputum  at  a  height  of  1356 
meters.  Treskinskaja  used  pure  cultures  emulsified  in  one-percent 
peptone  solution,  spread  in  thin  layers  and  exposed  directly  to  the 
sun.  These  were  killed  as  follows:  At  a  height  of  1560  meters, 
in  three  hours ;  at  903  meters,  in  four  hours ;  at  sea  level,  in  four 
hours.  The  peptone  solution  when  dried  gave  a  thin  protective 
layer  covering  these  organisms.  Treskinskaja  thought  that  this 
protection  would  be  about  equal  to  that  of  a  thin  layer  of  sputum. 


1QI2\  FATE  OF  TTBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  Bony  293 

The  difference  in  killing  power  is  due  to  the  difference  in  ultra 
violet  light.  These  rays  are  largely  absorbed  by  the  atmosphere, 
especially  when  laden  with  moisture.  According  to  Langley,  only 
39  percent  of  the  ultra  violet  light  reaches  the  sea  level.  The 
higher  the  altitude,  the  dryer  the  atmosphere,  the  more  intense  is 
the  light  and  the  greater  its  killing  power. 

Mitchell  and  Crouch  found  that  tubercle  bacilli  in  sputum  were 
not  killed  in  35  hours,  but  were  killed  in  45  hours.  These  experi- 
ments are  not  comparable  with  the  experiments  of  Treskinskaja, 
done  at  nearly  the  same  altitude,  since  the  soil  and  sputum  give 
far  more  protection  than  exposure  in  a  thin  layer  on  glass  from  a 
one-percent  peptone  solution. 

DISCREPANCIES  ^  's  not  to  ^e  exPected  that  tubercle  bacilli  under 
IN  RESULTS  different  conditions  will  be  killed  in  the  same 
length  of  time.  It  is  indeed  difficult  to  expose 
different  pure  cultures  under  the  same  conditions.  There  may  be 
a  difference  in  the  age  of  the  culture,  in  the  uniformity  of  the 
emulsion,  in  the  manner  of  exposing,  and  in  the  means  of  deter- 
mining the  time  of  killing  the  germs.  When  exposed  on  threads 
of  linen  or  silk  some  of  the  germs  may  be  well  protected  and  live 
for  a  much  longer  time  than  when  exposed  in  a  thin  layer  on  a 
glass  slip  or  on  sterilized  glazed  paper.  Then  if  the  material  in 
which  they  are  exposed,  as  sputum,  is  of  such  a  nature  as  to  give 
protection  from  drying  as  well  as  from  the  rays  of  the  sun,  the 
difference  will  be  still  greater.  This  accounts,  partly  at  least,  for 
the  difference  in  the  results  of  Sawitsky128,  which  showed  that 
tubercle  bacilli  in  sputum  on  linen  threads  lived  two  and  one-half 
months,  and  the  fact  reported  by  Weinzirl160,  that  a  pure  culture 
in  thin  layers  exposed  directly  to  the  sun  was  killed  in  a  few;  min- 
utes. It  is  not  possible  to  give  definite  reasons  for  all  the  discrep- 
ancies found  in  the  results  shown  in  Table  2. ;  but  excepting  those 
of  Feltz38,  whose  material  mixed  in  the  soil  might  well  give  com- 
plete protection  from  the  sun,  it  will  be  seen  that  tubercle  bacilli 
exposed  to  the  sunlight  are  killed  within  a. few  minutes  to  a  few 
hours. 

DIFFUSE  LIGHT  It  takes  ten  to  fifteen  times  as  long  for  tubercle 
bacilli  to  be  killed  in  diffuse  light  as  in  direct  sun- 
light; yet  these  germs  are  killed  much  sooner  in  diffuse  daylight 
than  in  the  dark,  when  under  the  same  conditions  otherwise,  as 
may  be  seen  by  comparing  the  results  of  Rickards,  Slack  and 
Arms119,  Twichell149,  and  Ransome  and  Delepine114. 

Tables  2.  and  3,  respectively,  give  in  brief  the  literature  upon 
these  subjects. 


294  BULLETIN  No.  161  [November. 

TABI,E  2. — EKKECT  OK  SUNSHINE  ON  TUBERCLE  BACILLI 


Author 

Date 

Conditions 

Not  killed 

Killed 

Feltz38 

Koch78 

Sawitsky128 
Ransome  and 
Delepine114 

Renzi117 
Migneco96 
Straus148 

Gardiner47 
Ottolenghi107 

Jousset" 

Mitchell  and 
Crouch98 
Abba  and 
Barelli1 
Annett4 

Cadeac18 
Bang11 

Twitche,!!149 
Didonna30 

Weinzirl160 

Rickards, 
Slack  and 
Arms119 

148 

Treskinskaja 

1890 

1890 
1891 
1894 
1894 
1895 
1895 

1898 
1899 

1900 
1900 
1900 

1901 
1902 

1903 
1905 

1905 

1905 
19U6 

1906 
1909 

1910 

Sputum  and  soil  mixed  and  exposed 
to  direct  sunlight,  tested  by  gui- 

137  days 
2%  mo. 

6  hrs. 
10  to  15  hrs. 

1  to  12  hrs. 
24  hrs. 

1  hr. 
35  hrs.1 

4.76  percent 
2  to  24  hrs. 

24  hrs. 
1  hr. 

2  to  8  hrs.2 
6  hrs. 

after  137 
days 
after2>£mo. 
few  min.  to 
few  hrs. 
2y2  mo. 

45  days 
4  days  (12# 
hrs.  sun) 

24  to  30  hrs. 
2  hrs. 

40  hrs. 
24  to  30  hrs. 
9  hrs. 
6  hrs. 

4  to  14^  hrs. 
5  to  7  hrs. 
1  hr. 

45  hrs. 
6  days 

48  hrs. 
24  hrs. 
48  hrs. 

6  min. 
7  hrs. 

2  to  10  min. 

3  hrs. 
4  hrs. 

4  hrs. 

Exposed  to  changing  climatic  condi- 

In  pure  culture     

Sputum  on  stretched  linen  

Sputum  exposed  to  light  

Pure  culture  on  glazed  paper  

Sputa  mixed  with  10  parts  water,  ex- 
posed at  28°  C  

Sputum  on  stretched  linen  and  woolen 
threads  

Culture  in  a  glass  container,  in  broth 
In  a  thick  layer  of  dried  sputum  

In  sputum  on  woolen  cloth  

Sputum  the  thickness  of  spitting,  on 

Sputum  exposed  to  direct  sun  

Tuberculous    sputum     placed     upon 

Tested  105  specimens  of  sputa  taken 
at  random;  5,  or  4.  76  percent  con- 

Tuberculous  sputum  size  of  spitting. 
Tuberculous  sputum  on  a  board  

Tuberculous  sputum  on  glass  

Tubercle  bacilli  on  agar,  exposed  to  a 
30-ampere  lamp  at  30  cm.  distance 
Tuberculous  sputum,  direct  rays  
Exposed  culture  to  sun  2  to  8  hours; 
then   inoculated   guinea    pigs    in 
order   to   immunize   them.       Pro- 
duced only  a  local  abcess  

Pure  culture  on  paper  slips  or  glass 

Sputum  exposed  in  sunshine     

Summer  months,  direct  sun: 
Pure  culture  tubercle  bacilli,  height 
1560  m    

Pure  culture  tubercle  bacilli.  903  m 
Pure  culture  tubercle    bacilli,    sea 
level  ...          

Virulence  diminished  after  20  hours. 

2It  is  not  certain  whether  the  organisms  were  alive  or  dead,  since  dead  organ- 
isms can  produce  tubercles. 


igi2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  295 

TABLE  3 — ACTION  OK  DIFFUSE  LIGHT  UPON  TUBERCLE  BACILLI 


Author 

Date 

Conditions 

Not  killed 

Killed 

Galtier46 

1889 

Tuberculous  organs: 
Dried  at  30°  

38  days 

30  days 

Dried  at  room  temperature  

(some)  30 

(usually)  af- 

Sawitsky128 

1892 

Sputum  on  stretched  linen  

days 

ter  30  days 
2%  mo. 

Ransome  and 
Delepine1  ^  * 

1894 

Tubercle  bacilli  on  dried  paper  

2  days 

lyucibelli89 

1899 

Sputum  dried  on  glass    

18  days 

Fluid  sputum  in  reagent  glass  

4  mo 

Jousset66 

1900 

4  to  7  hrs 

1902 

8  to  14  days 

Mixed  with  dust   

8  days 

On  threads      

10  days 

On  coarse  cloth  

20  days 

Hill80 

1903 

Sputum  in  reagent  glass  in  glass  cup- 

16  days 

Cadeac1  8 

1905 

2  to  6  days 

4  to  10  days 

Twitchell149 

1905 

Tuberculous  sputum  in  paraffined  bot- 
tles    

124  days 

175  days 

Sortnani1  38 

1906 

Dry  sputum  in  a  room  

1  mo 

Rickards, 
Slack  and 
Arms119 

1908 

Dry  sputum  in  a  room   

1  mo 

Weinzirl159 

1908 

Fine  emulsion  of  tubercle  bacilli  dried 
on   paper   slips    (average   of    ten 
trials)     .'. 

2  8  days 

4.4  days 

EFFECT  OF  DESICCATION  UPON  TUBERCLE  BACILLI  IN  SPUTUM 
AND  OTHER  MATERIAL 

The  difference  in  the  length  of  time  that  different  bacteria 
withstand  desiccation  is  very  great.  B.  cartovorus  is  killed  in  a 
few  minutes  to  a  few  hours  (Jones64)  ;  B.  tuberculosis  may  be 
killed  in  a  few  days  ( 14  days,  Maffucci90)  to  several  days  (88  days, 
Rickards119);  and  spores  of  Bad.  anthracis  live  twelve  years 
(reported  above). 

The  difference  depends  upon  the  kind  of  substance  on  which 
they  are  exposed  and  upon  the  difference  in  the  kind  and  form  of 
organism  exposed.  Harding  and  Prucha54  have  shown  that  Bact. 
cauipcstre  remains  alive  much  longer  when  dried  on  cabbage  se^d 
than  when  dried  on  glass  covers;  on  glass  it  was  dead  at  the  end 
of  ten  days;  on  the  seed  it  remained  alive  for  thirteen  months. 
This  difference  is  no  doubt  largely  due  to  the  difference  in  the 
hygroscopic  moisture  retained  by  these  substances.  The  kind  and 
form  of  the  organism  exposed  to  drying  has  even  more  to  do  with 
its  capability  of  living.  The  spore  form  lives  very  much  longer 
than  the  vegetative  form. 


296  BULLETIN  No.  161  [Xorembcr, 

It  appears  that  tubercle  bacilli,  especially  in  sputum  and  other 
mucoid  material,  withstand  desiccation  better  than  other  nonspore- 
formers.  The  difference  is  not  great  and  there  are  many  apparent 
exceptions  where  other  nonspore-bearers  live  for  a  very  long  time, 
like  the  one  given  aboVe  after  Harding  and  Prucha54.  Diplococcus 
pneumonia  has  been  reported  as  living  for  192  days  when  dried 
on  silk  (Sirena  and  Alessi135). 

The  important  fact  which  has  been  so  thoroly  established  in 
recent  years  is  that  tuberculous  sputum  reduced  to  dust  causes 
tuberculosis  when  experimental  animals  (guinea  pigs,  rabbits,  cats, 
dogs  and  calves)  are  made  to  breathe  such  dust-laden  air.  How 
great  the  danger  is  to  man  and  cattle  to  breathe  dried  tuberculous 
material  is  yet  a  disputed  question.  In  1882  Koch,  in  his  work 
on  "Die  Aetiologie  der  Tuberculose",  pointed  out  that  dried  tuber- 
culous sputum  is  one  of  the  most  important  factors  in  causing 
tuberculosis.  It  was  not  until  1905  when  the  work  of  Calmette 
and  Guerin21  was  published  that  ingestion  was  brought  forward 
as  a  very  important  factor  in  producing  tuberculosis.  It  was  sug- 
gested by  the  advocates  of  this  theory  that  practically  all  tubercu- 
losis is  produced  by  ingestion — that  even  inspired  material  is  swal- 
lowed and  what  apparently  is  inhalation  tuberculosis  is  actually 
tuberculosis  by  ingestion.  This  conclusion  will  not  hold  in  the 
light  of  the  most  recent  experiments,  especially  those  carried  out 
at  Breslau  and  collectively  published  by  Fliigge40  (1908),  "Ver- 
breitungsweise  and  Bekampfung  der  Tuberknlose".  It  is  shown 
here  that  in  all  animals  with  which  experiments  have  been  made 
it  required  a  hundred  to  a  thousand  times  more  tubercle  bacilli  to 
produce  tuberculosis  by  feeding  than  it  does  by  inhalation.  So  the 
thought  that  the  swallowing  of  a  part  of  the  few  tubercle  bacilli 
necessary  to  produce  tuberculosis  by  inhalation  is  the  cause  of  in- 
gestion tuberculosis  is  wholly  precluded. 

A  complete  discussion  of  this  most  interesting  subject  cannot 
be  given  here.  The  data  in  the  two  following  tables  show  the  fact 
mentioned  above,  that  it  requires  many  times  more  germs  to  pro- 
duce'tubercnlosis  by  feeding  than  by  inhalation ;  also  another  most 
important  fact,  that  severe  tuberculosis  may  be  produced  by  either 
method  of  infection.  It  must  not  be  understood  that  the  facts 
exhibited  in  these  two  tables  in  any  way  indicate  which  is  the 
more  frequent  method  of  infection.  For  what  matter  if  it  does 
require  one  thousand  times  more  tubercle  bacilli  to  produce  tuber- 
culosis by  feeding  than  by  inhalation,  who  knows  whether  we  are, 
on  the  average,  taking  in  one  thousand  times  more  of  these  germs 
in  the  food  than  in  the  breath  ? 

On  one  hand,  Calmette19  is  found  declaring  that  "In  the  ordi- 
nary daily  life,  the  infection  of  the  digestive  organs  is  predomi- 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


297 


Remarks 

erms  inhaled  in  moist  spray 
thru  a  tracheotomy  tube, 
erms  inhaled  as  above.  All 
became  severely  tubercu- 
lous. 
11  except  3  of  the  65  guinea 
pigs  became  tuberculous; 
two  that  had  received  40 
and  one  of  the  three  that 
had  received  20  individual 
germs  remained  healthy. 

he  guineapigs  in  each  series 
were  killed  1,  12,  24,  72  and 
144  hours  after  inhalation. 

11  became  tuberculous. 

ses  the  Findel  tower  ap- 
paratus for  inhalation  in 
the  three  series, 
nly  doses  of  50,000  germs 
surely  positive  in  the  last 
series. 

he  dose  was  given  with  the 
Buchner  spray  apparatus  in 
the  open  air;  so  only  a  part 
was  inhaled. 

nimals  inhaled  tuberculous 
sputum  dried  12  to  13  days 
on  a  carpet;  beaten  and 
swept  for  20  minutes, 
ame  as  above  except  dried 
for  6  days  and  beaten  and 
swept  for  12  minutes, 
uinea  pigs  exposed  during 
the  time  of  the  above  two 
series  and  also  the  time  of 
removing  the  first  and  plac- 
ing the  second  series  of 
test  animals. 

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298 


BULLETIN  No.  161 


[November, 


Remarks 

uckled  mothers  whose  teats 
were  injected  with  pure 
culture  of  tubercle  bacilli, 
ame  as  above, 
ed  thru  a  stomach  tube, 
he  human  type  of  tubercle 
bacilli  appeared  not  to 
injure  the  goats.  In  June 
they  were  given  200  mg.  of 
bovine  type. 
11  severely  tuberculous: 
lung  involvement. 

11  became  tuberculous. 

ix  Breton  heifers  aged  8  to 
10  months. 
11  became  tuberculous. 

hree  were  fed  at  three  in- 
tervals during  40  days; 
other  two  at  one  feeding, 
wo  control  guinea  pigs  in- 
oculated with  19,000  indi- 
vidual tubercle  bacilli  be- 
came severely  tuberculous. 

o  tuberculosis  in  any  ani- 
mal, 
nly  slight  evidence  of  tu- 
berculosis in  one  that  was 
fed  50  mg. 

ot  the  slightest  evidence  of 
tuberculosis  in  any  animal. 

nlyone  calf  was  completely 
negative.  The  presence  of 
the  disease  was  found  21 
times  in  the  abdominal  or- 
gans, but  only  3  times  in 
the  lungs.  Always  present 
in  the  abdominal  organs 
when  found  in  the  lungs. 

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FATK  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


299 


nant";  and  on  the  other  hand  Chausse'j:  says,  "Tuberculosis  in 
adult  bovine  is  due  to  inhalation  in  98  percent  of  the  cases,  if  not 
more;  in  calves  in  about  90  percent,  ingestion  and  congenital  in- 
fection being  responsible  for  the  remainder."  Dr.  Ravenel115,  a 
strong  believer  in  ingestion  as  one  of  the  important  causes  of 
tuberculosis  says,  "One  must  take  an  impartial  view  of  the  whole 
problem,  and  be  willing  to  agree  that  both  channels  of  infection 
are  open.  In  animals,  however,  the  alimentary  tract  seems  to  be 
a  more  common  port  of  entry". 

At  present,  at  least,  both  ways  of  infection  should  be  consid- 
ered very  dangerous  and  every  precaution  taken  to  guard  against 
them.  A  brief  summary  of  the  literature  concerning  the  length  of 
time  that  tubercle  bacilli  live  in  dried  tuberculous  material  and 
concerning  the  infectiousness  of  such  material  is  given  in  Table  6. 

TABI.E  6 — EFFECT  OK  DESICCATION  OF  TUBERCLE  BACILLI  IN  SPUTUM  AND 

OTHER  MATERIAL 


Author 

Date 

Conditions 

Not  killed 

Killed 

Villemin153 

1869 

Dried  tuberculous  sputum  

several  hrs. 

Koch74 

1882 

8  wks. 

Cochez2* 

Malassez  arid 
Viffnal91 

1883 
1883 

Tubercle  bacilli  incased  in  sputum  
Dried  sputum  killed  a  dog,  by  inhala- 
tion 
Alternate  drying  and  moistening  of 
tuberculous  sputum  8  times.  ...... 

3  wks 
12  days 

Schill  and 
Fischer1*9 

1884 

Tuberculous    sputum    with    spores, 
dried  on  glass  

126  days 

179  days 

Tuberculous  sputum  without  spores, 

186  days 

226  days 

DeThoma89 

1886 

10  mo. 

Sormani137 

1886 

Dried  tuberculous  sputum  

2  mo. 

virulence 

Galtier46 

1887 

Tuberculous  sputum  dried  on  linen.  .  . 
Dried  tuberculous  material  

6  mo. 
20  to  38  days 

deceased 
thereafter 
no  virulence 
after  4  mo. 

Cadeac  and 
Malet17 

1888 

Pieces  of  dried  tuberculous  lung  ex- 
posed on  paper  in  laboratory  

43  days 

102  days 

Dried  tuberculous  lung  allowed  to  de- 

76  days 

80  days 

Same  repeated  

150  days 

after  150 

De  Souza28 

1888 

Inhalation  of  dried  tuberculous  mate- 
rial caused  tuberculosis  in  12  of  14 
guinea  pigs.      Time  of  drying  not 

days 

Galtier46 

1888 

Inoculation   of    tuberculous   material 
dried    at   30°  C.,  or  by  breathing 
dried  tuberculous  material  

15,  30  and 

Same  material  dried  at  room  temper- 
ature    

38  days 
30  days 

after  30 

days 

300 


BULLETIN  No.  161 
TABLE  6 — Continued 


\Xorcinbcr. 


Author 

Date 

Conditions 

Not  killed 

Killed 

Cornet" 
Feltz38 

Sawitsky128 

Stone140 
Koch72 

Maffucci80 

Marpmann92 

Ransome  and 
Delepine114 

Hance83 
Kirchner68 

1889 
1890 

1891 

1891 
1892 

1892 

1893 
1894 

1895 

1895 

1 

Dust   samples  examined   for  tubercle 
bacilli  were  found  positive: 
In  hospitals  

17.6  percent 
17.6 
43.6         " 

over  7  mo. 
about  140 
days 
7  to  9  mo. 

2%  mo. 
2}4  mo. 

3  yrs. 

2  to  8  wks. 
14  days 

2  mo.  (?) 
(?) 

19  days 

19  days 
45  days 

4.9  percent 
25  percent 

2  mo.  (?) 
19  days 

all  killed 

In  insane  asylums   

In  dwelling1  houses  

)ried  tuberculous  sputum  in  road  dust: 
Exposed  to  weather.  

Exposed  to  sun   •  •>••• 

Dried  sputum   in  rooms  

Tuberculous   sputum  dried  under  or- 
dinary conditions  and  exposed  in 
the  dark  in  living"  rooms  

Same  exposed  to  sunlight  

Dried   tuberculous  sputum,  only  de- 

Guinea    pigs   were    inoculated    with 
tuberculous    sputum   dried    for  2 
weeks,    for    4   weeks,    and    for   8 
weeks.     In  each  case  tuberculosis 
developed  as  with  fresh  material. 
3ure  culture  of  avian  tubercle  bacill 
dried   on  silk  threads,  inoculated 
into  a  hen  abdominally  

Same  repeated.     Hen  not  tuberculous 
abdominally;  one  tubercle  in  the 

Tubercle   bacilli  frequently  found  in 
street  dust  by  microscopic  test  .  .  . 

Tuberculous  sputum  exposed  in  watch 
glasses: 
To  air   and  light  4  days;    then   15 

To   air  and  light  8  days;    then  11 
days  in  the  dark  

In  closed  cupboard  

In   dark   air   shaft,  produced  slight 
tuberculosis  in  a  rabbit  

The   author,    under  the  direction  of 
Dr.   Trudeau   of   the   Adirondack 
Cottage  Sanitarium,  examined  81 
samples  of  dust  from  the  differ- 
cottages.     Four  guinea  pigs  dice 
too  early  to  make  the  test  for  tu 
bercle  bacilli;  of  those  remaining 
5,  or  4.9  percent,  became  tubercu- 
lous.     These  were  from  a  group 
of  10  guinea  pigs  inoculated  with 
the  dust  from  one  small  cottage. 
The     author    later    inoculated    9 
guinea  pigs  with  dust  from  a  New 
York  hospital,  5  of  which  died  in 
3  days,  and  1  of  the  4  remaining 
became  tuberculous  

Fourteen  guinea  pigs  were  inoculated 
with  7  samples  of  dust  from  rooms 
in    homes    of    consumptives;     9 
guinea  pigs  died  early  and  5  re- 
main^d  healthy.    In  a  second  ser- 

FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 
TABLE  6 — Continued 


301 


Author 


Date 


Conditions 


Not  killed 


Killed 


Kirchner  es 
(continued) 


Cornet2 


Laschtsch- 
enko78 

Neisser102 


Beninders 


1893 

1898 
1898 

1899 


Cornet26 


Sticker139 


Jones* 


1899 


1899 


1900 


Peterson1 


Heymanu59 


1900 


1901 


ies  three  rooms  were  investigated, 
from  which  8  guinea  pigs  were  in- 
oculated; one  died  early  and  one 
of  the  other  7  became  tuberculous. 

Tuberculous  sputum  placed  on  a  car- 
pet and  allowed  to  dry  naturally 
produced  tuberculosis  in  35  out  of 
36  guinea  pigs  which  were  placed 
in  a  room  at  a  distance  of  134  to 
300  cm.  from  this  carpet 

Tuberculous  sputum  dried  in  thick 
layers  on  paper  and  cloth  was  not 
virulent  after  9  months 

Tuberculous  sputum  rubbed  up  with 
dust  and  aspirated  at  a  velocity 
of  3  to  5  cm.  per  second  will  pro- 
duce tuberculosis,  when  inocu- 
lated into  guinea  pigs,  in  a  large 
majority  of  cases 

Handkerchiefs  that  were  used  by  con- 
sumptives from  2  to  12  hours  were 
dried  for  24  hours,  and  then  rubbed 
up  in  a  box.  The  dust  from  these 
was  aspirated  and  injected  into  two 
guinea  pigs  for  each  t'est.  In  12 
tests  8  were  positive  and  4  nega- 
tive  

Dried  sputum  in  one  case  lost  viru- 
lence in  3  months;  another  time 
remained  virulent  from  6  to  8 
months 

Tested  29  samples  of  sputum  dried  from 
2  to  21  days,  by  inoculating  guinea 
pigs  with  dried  sputum  dust  and 
by  causing  them  to  inhale  the 
dried  sputum  dust.  Tuberculosis 
was  produced  in  27  out  of  29  tests. 

The  author  inoculated  guinea  pigs 
interperitoneally  with  the  mucus 
from  the  nostrils  of  131  healthy 
persons.  Three  of  these  guinea 
pigs  died  of  tuberculosis  on  the 
8th,  14th  and  59th  days  after  in- 
oculation. No  tubercle  bacilli 
could  be  found  b}r  stains  of  the 
nasal  mucus' 

One  of  8  guinea  pigs  became  tuber- 
culous after  inhaling  sputum, 
dried  for  2  months;  while  sputum 
dried  for  3  months  gave  one  posi- 
tive in  6 

The  aspirated  air  from  sputum,  dried 
6  days  and  injected  into  guinea 
pigs,  produced  tuberculosis 


14  percent 


9  mo. 


8  positive 


8  mo. 


Positive 
27  of  29 
tests 


4  negative 


2  mo. 

3  mo. 

6  days 


'It  is  probable   that   these  three  guinea  pigs,  or  at  least  the  first  two,  died  of 
spontaneous  tuberculosis. 


302 


BULLETIN  No.  161 
TABLE  6 — Continued. 


[November, 


Author 


Date 


Conditions 


Not  killed 


killed 


Heymann51* 
(continued) 


Gotschlich" 


Hill60 


1903 


1903 


Noetel105 


1904 


Cadeac18 


Kirstein69 


1905 


1905 


Dust  from  rooms  of  consumptives: 
Samples  taken  on  a  brush 

No.  of     No.  of     Percent 
samples  positive  positive 
Private  rooms 59  5  8.5 

Hospitals          61  5  8.2 

Samples  taken  on  a  moist  sponge 
Private  rooms  57  9  15.78 

Hospitals         62  25  40.3 


Total  sa'ples  239  44  18.4 

Hospitals        123  30  24.3 

Pri'te  homes  116  14  12.0 

No  tubercle  bacilli  were  found  in  119 
samples  of  dust  taken  from  15 
places  where  consumptives  were 
in  the  habit  of  visiting. 
Samples  of  tuberculous  sputum  from 
10  rooms,  using  496  swabs,  were 
.taken  by  rubbing  them  on  carpet, 
furniture,  etc.  In  8  of  the  10 
cases  the  sputum  was  proven  to 
contain  tubercle  bacilli  from 
stained  preparations;  the  other 
two  were  not  tested.  Tubercle 
bacilli  dried  on  glass  rods  were 
dead  after  16  days. 

Clothing  that  had  been  and  that  was 
being  worn  daily  by  consumptives 
was  enclosed  in  a  large  box  of 
three  cubic  meters  content.  The 
dust  was  beaten  and  shaken  out 
and  allowed  to  settle;  then  it  was 
collected  and  thoroly  rubbed 
up  in  5  cc.  of  broth.  The  original 
material,  and  dilutions  of  .1  and 
.01  were  respectively  injected 
into  guinea  pigs: 

Coat  and  vest  worn  daily 

Jacket  and  hose  worn  daily 

New  jacket,  hose  and  old  vest  with 
no  evidence  of  contaminated 
sputum 

Old  coat 

Coat,  hose  and  plush  vest,  none 
worn  for  three  weeks 

Wool  jacket  and  old  hose,  not  worn 

for  five  weeks 

Tuberculous  sputum  exposed  on  glass 

to  air  and  light 

Tuberculous  sputum,  in  thick  layers 

dried  on  a  marble  slab 

Tubercle  bacilli  from  pure  culture  or 
from  tuberculous  sputum  were 
sprayed  upon  collected  dust,  and 
then  after  different  lengths  of 
time  tested  for  virulence  by  inoc- 
ulation of  guinea  pigs.  Dust  from : 


Positive 
Positive 


Negative 
Positive 

Positive 
Positive 

2,  4  and  6dys 


4,6andlOdys. 
14  days 


1<)I2\  FATE  OF  TI.T.ERCI.E  BACILLI  OUTSIDE  THE  ANIMAL  BODY  303 

TABLK  6. — Continued 


Author 


Kirstein1"'1' 
(continued) 


Park  and 

Williams110 

Twitchell1*9 


Sormani138 
LeNoir  and 
Camus88 


Rodet  and120 
Delanoe 


KShlisch71 


Datt- 


1905 
1905 


1906 
1907 


1907 


1908 


Conditions 


Books  and  papers 

Sputum  dust 

Cloth  ravelings 

Street  dust 

Dried  tuberculous  sputum    gradually 
loses  its  virulence,  but  still  infective 
Tuberculous  sputum: 

In  paraffined  bottles  in  dark  moist 
box 

In  paraffined  bottles  in  diffuse  light 

In  paraffined  bottles  in  thermostat. 

In  cotton-stoppered  bottles  in  dark 
moist  box 

In  cotton-stoppered  bottles  in  dark 
closet 

On  ice 

Tuberculous  sputum: 

On  handkerchiefs.   

On  towels 

On  carpets  

In  sand  in  moist  light  place 

In  sand  in  dry  light  place 

In  open  bottles,  out  doors,  winter  .  . 
Dried  tuberculous  excretion  at  35°  C. 
Ivarge  quantities  of  air  were  aspirated 
from  tuberculous  hospitals  and  the 
dust   injected   under  the  skin  of 
guinea  pigs.     From  4  tests  aspi- 
rating respectively  270,  2000,  20000 
and   53000  liters,  in  no  case  were 

tubercle  bacilli  found 

Eleven  samples  of  dust  from  local 
offices  and  private  dwellings  were 
inoculated  into  guinea  pigs,  with 

wholly  negative  results 

Fresh  tuberculous  sputum  or  a  pure 
culture  of  tubercle  bacilli  was  inti- 
mately mixed  with  dust  obtained 
from  dwellings  and  factories,  and 
the  number  of  tubercle  bacilli  that 
a  guinea  pig  inhales  in  a  given 
time  was  determined.  It  was 
found  that  tuberculosis  was  pro- 
duced in  the  test  animals  when  as 
low  as  50  tubercle  bacilli  were  in- 
haled, and  that  the  inhalation  of 
270  or  more  of  these  germs  al- 
ways produced  tuberculosis.  An- 
other series  of  experiments  were 
made  with  dust  from  15  dwellings 
where  consumptives  were  living. 
The  rooms  were  small  and  poorly 
kept.  F/ighteen  guinea  pigs 
breathed  for  some  time  in  dense 
clouds  of  this  dust.  None  of  these 
guinea  pigs  became  tuberculous. 


8  days 

4  days 

5  days 
3  days 

2  to  3  mo. 


170  days 

124  days 

33  days 

157  days 

100  days 
102  days 

70  days 

70 

39 
123 

30 
110 

15  days 


Not  killed        Killed 


14  days 

7  days 
10  days 

8  days 


188  days 
175  days 
100  days 

172  days 

141  days 
153  days 

110  days 
110 

70 
148 

70 
132 


all 


11  samples 


304 


BULLETIN  No.  161 
TABLE  6 — Continued 


[November, 


Author 


Date 


Conditions 


Not  killed 


Killed 


Kuss1 


1908 


Tuberculous  sputum  dried  in  thin 
layers  in  the  dark,  free  to  the  air: 

Virulence  wholly  preserved 

Viruletice  diminished 

Virulence  much  decreased,  yet  a 
large  dose  produced  a  tuberculous 
guinea  pig 

Virulence  entirely  lost 

Tuberculous  sputum,  dried  in  thin 
layer,  in  diffuse  light  of  a  room 
rapidly  decreased  in  virulence: 

Noticeable  decrease 

Marked  decrease 

Very  noticeable  decrease 

Not  entirely  lost 

Entirely  lost 

Two  guinea  pigs  were  exposed  to  the 
dust  made  by  shaking  of  hand- 
kerchiefs on  which  tuberculous 
sputum  had  been  dried  for  25  days. 

Neither  became  tuberculous 

Six  guinea  pigs  were  exposed  to  the 
dust  from  a  plank  containing 
sputum  that  had  been  drying  for 

17  days.  Remained  healthy 

Experiments  in  a  box  of  125-liter  con- 
tent: 

Tuberculous  sputum  dried  for  6  days 
in  the  dark  was  powdered  and  1 
to  2  grams  suspended  in  the  air  of 
this  box.  Guinea  pigs  with  their 
heads  projecting  into  this  box 
were  made  to  breathe  this  dust 
from  30  to  60  minutes.  In  all  cases 
the  guinea  pigs  became  tubercu- 
lous  

Experiments  in  a  room  of  30  cubic 
meters: 

Series  i — 125  cc.  of  tuberculous 
sputum  were  dried  on  a  carpet 
from  11  to  13  days.  Nine  guinea 
pigs  were  swung  60  to  90  inches 
above  this  carpet.  Then  the  car- 
pet was  beaten  and  swept  for  20 
minutes.  Four  of  the  9  guinea 
pigs  became  tuberculous.  Also 
the  dust  obtained  by  the  aspira- 
tion of  60  liters  of  air,  at  a  point 
60  inches  above  this  carpet,  was 
inoculated  into  a  guinea  pig, 
which  became  severely  tubercu- 
lous   

Series  2. — 20  cc.  of  tuberculous  spu- 
tum were  dried  on  another  carpet 
for  6  days.  Six  guinea  pigs  were 
suspended  above  this  carpet, 
which  was  beaten  and  swept  for 
12  minutes.  Two  of  the  6  guinea 
pigs  became  tuberculous.  Also 


12  to  14  days 
18  days 


20  to  30  days 


3  days 

7  days 

10  days 

15  days 


40  to  50  days 


20  days 

25  days 
15  days 


6  days 


11  to  13 


I9I-]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 

TABLE  f> — Continued 


305 


Author 


Date 


Conditions 


Not  kilk-d         Killed 


Kuss77 
(continued) 


LeNoir  and 
Camus83 


1908 


1908 


L/eNoir  and 
Camus86 


1908 


dust  obtained  by  the  aspiration  of 
71  liters  of  air  produced  a  tuber- 
culous guinea  pig 

Series  3 — Six  of  the  9  guinea  pigs 
exposed  to  the  beatings  and 
sweepings  of  the  two  carpets  of 
Series  1  and  2,  described  above, 

became  tuberculous 

Tuberculous  sputum  rich  in  tubercle 
bacilli  was  mixed  with  dust  a  part 
of  which  had  been  sterilized, 
another  part  not  sterilized,  and 
each  mixed  sample  was  dried  in  a 
large  open  flask  and  kept  in  the 
laboratory  for  33  days.  Each  sam- 
ple was  inoculated  into  a  series  of 
5  guinea  pigs: 

Series  i.— Dust  not  sterilized.  Three 
of  the  5  guinea  pigs  became  tu- 
berculous only  in  the  lymphatic 
glands  near  the  point  of  inocula- 
tion1   

Series  2 — Dust  sterilized.  Three  of 
the  5  guinea  pigs  died  of  acute  in- 
fection; the  other  2  had  tubercu- 
lous lymph  glands  near  the  point 
of  inoculation,  and  one  of  the  two 

had  a  tubercle  in  one  lung1 

Again,  the  author  examined  dust 
from  a  tuberculous  hospital: 

Series  3. — The  dust  was  collected 
from  the  cornice  near  the  ceiling, 
wash-board,  window-sill,  window- 
seat,  bed  railings  and  floor.  All 
was  thoroly  mixed,  divided  into 
two  lots  of  .6  gram  each,  and 
placed  in  two  flasks.  One  flask 
was  protected  from  the  light;  the 
other  was  exposed  to  diffused 
light  for  5  days  and  to  sunlight  3 
days.  Each  sample  was  inocu- 
ulated  into  a  series  of  5  guinea 
pigs.  In  each  case  only  one  of 
the  5  test  animals  became  tuber- 
culous. 

Series  4. — Four  grams  of  dust  were 
collected  and  treated  as  in  Series 
1.  In  this  series  2  of  the  guinea 
pigs  died  of  acute  infection  and 
3  remained  healthy. 

Series  5. — Dust  was  collected  and 
treated  as  in  Series  1.  0.7  grams 
of  the  dust  was  placed  in  each  of 
two  flasks.  The  first  was  exposed 


6  days 


6  to  13 
days 


33  days 


33  days 


JThe  tuberculosis  may  have  been  produced  from  dead  tubercle  bacilli. 


306 


BULLETIN  No.  161 
TABLE  §.— Continued 


\  November, 


Author      xj  Date 

Conditions 

Not  killed 

Kilk-d. 

L<eNoir  and 
Camus815 

Verdozzi152 

Rickards, 
Slack 
and 
Arms119 

t 

LeNoir 
and 
Camus8  2 

1908 

1909 
1909 

1909 

for  10  days  to  the  sun;  and  the 
second  in  the  shade  for  the  same 
time.  A  series  of  5  guinea  pig's 
was  inoculated  from  each  sample. 
In  each  case  4  of  the  test  animals 
died  of  acute  infection  within  a 
few  days;  the  other  lived  17  days 
and  then  died  of  an  unknown  dis- 
ease. Neither  became  tubercu- 
lous   

3  to  10  days 
10  to  14  days 
6  hrs. 

19  days 
31  days 

35  days 

65  days 
88  days 

10  days 

after   19    to 
57  days 

25,     29,     32 
days    and 
afterward 

36  to65cla3Ts 

The  nasal  cavities  of  non-tuberculous 
persons,    living    in    a    room    with 
consumptives,  were  washed  with 
wet   cotton    swabs    and    this    ma- 
terial inoculated  into  guinea  pigs. 
Of  9  guinea  pigs  thus  inoculated 
2  died  with  abscesses  and  the  other 
7  remained  healthy. 
Further  investigations    were   carried 
out  on  consumptives.   Of  13  guinea 
pigs  inoculated  3  showed   tuber- 
culous changes.1 
Dust  from  the  library  at   Rome  pro- 
duced tuberculosis  in  rabbits. 
Tuberculous  sputum    was   placed   on 
small  strips  of  carpet  and  wooden 
tongue  depressors  and  exposed  in 
tenement  houses  in  Boston: 
To  diffuse  daylight     

jn  the  dark       

To  the  sunlight  

The    same    exposed    in    the    Boston 
Health  Laboratory: 
In  diffuse  daylight,  on  carpet.  
In  diffuse  day  light,  on  wood: 

In  the  dark  exposed  in  the  air  on 
carpet: 

Negative  then  till  65  days  

At  this  time  (65  days)  two  speci- 
mens rubbed  together  were  posi- 

On  wood  no  end  point  was  reached. 
Samples  were  positive  up  to   -  •  • 
Ten  rabbits  in  one   box  and  4  in  an- 
other were  placed  in  a  tuberculous 
hospital,    and    arranged    so    that 
they  could  breathe  only  dust-laden 
air  from  the  rooms  with  tubercu- 
losis patients.    Four  of  the  10,  and 
2  of  the  4  rabbits   were  found  to 
be  tuberculous  when  examined  6 
weeks  later. 

'Tuberculosis    in    these   cases    was    more    likely^    produced    by    fresh    tubercle 
bacilli  from  the  patient  than  from  dust  breathed  by  the  patient. 


FATE  OF  TTUERCLE  BACILLI  OrrsinE  THE  ANIMAL  Bony  307 

TUBERCLE  BACILLI  IN  WATER  AND  OTHER  MATERIAL 

^act  t^lat  tubercle  bacilli  live  and  remain  viru- 


INTRODUCTORY 

STATEMENT        lent  in  water  for  so  long  a  time   (one  year  and 

more),  together  with  the  other  well-established 
fact  of  the  danger  of  the  ingestion  of  these  germs,  makes  their 
presence  in  drinking  water,  in  food,  and  in  the  soil  assume  special 
significance.  Tubercle  bacilli  gain  entrance  .to  water  supplies  thru 
dejecta  from  tuberculous  farm  animals,  especially  dairy  cattle,  as 
well  as  from  tuberculous  people  ;  in  the  latter  case  both  from  their 
dejecta  and  from  their  sputa.  With  these  facts  before  us  we  can 
well  conceive  the  danger  of  tuberculous  infection  from  drinking 
water  and  the  benefit  that  comes  from  a  purified  water  supply. 

MILLS-RE  i  NCKE  ^le  epidemics  of  typhoid  fever  which  at  times 
PHENOMENON  have  caused  the  death  of  hundreds  of  people  in  a 
city  within  a  few  weeks  have  frequently  been 
traced  to  the  water  supply.  This  brought  about  the  purification  of 
the  city  water  supplies.  Besides  the  lessening  of  typhoid  fever  in 
a  city  having  a  purified  water  supply,  there  has  been  noted  also 
great  benefit  from  the  lessening  of  other  diseases.  This  was  ob- 
served independently  by  J.  J.  Reincke,  of  Hamburg,  Germany,  and 
by  Hiram  F.  Mills,  of  Lawrence,  Massachusetts.  Each  was  the 
health  officer  of  his  respective  town.  This  fact  was  styled  by 
Sedgwick  and  MacNutt133  the  Mills-Reincke  phenomenon  —  "For 
every  life  saved  from  typhoid  fever  by  the  purification  of  a  water 
supply,  a  certain  number  of  lives  are  saved  from  other  diseases." 

The  other  diseases  more  especially  concerned  are  infant 
diarrhea,  tuberculosis,  and  bronchial  troubles.  Mr.  Allen  Hazen, 
at  the  International  Engineering  Congress  held  at  the  St.  Louis 
Exposition  in  1904,  gave  a  mathematical  equivalent  to  this  phenom- 
enon in  what  is  now  known  as  Hazen's  theorem,  viz.,  "Where  one 
death  from  typhoid  fever  has  been  avoided  by  the  use  of  better 
water,  a  certain  number  of  deaths,  probably  two  or  three,  from 
other  causes  have  been  avoided."  Sedgwick  and  MacNutt  found 
the  Mills-Reincke  phenomenon  and  Hazen's  theorem  to  be  sound 
and  conservative  when  vital  statistics  of  Lowell,  Albany  and  Bing- 
hampton  were  carefully  studied. 

Dr.  J.  J.  Reincke,  health  officer  of  Hamburg,  in  his  report  of 
1893,  nas  tne  following  to  say  concerning  the  lessening  of  tuber- 
culosis : 

"Especially^  surprising  was  the  improvement  (as  to  phthisis)  in  the  per- 
iod from  the  fourth  to  the  sixth  decade,  when  the  sewage  and  the  water- 
supply  system  just  completed  must  have  been  of  very  strong  influence  for 
general  cleanliness."  (Page  285). 


308  BULLETIN  No.  161  [November, 

"It  is  worthy  of  nptice  that  it  is  maintained  from  the  surgical  side  that 
since  the  filtration  of  the  water  the  number  of  cases  of  Hone  and  joint  tu- 
berculosis has  diminished  extraordinarily.  Further  observations  should 
tell  whether  a  causal  connection  really  exists  here.  At  any  rate  such  a 
possibility  cannot  be  excluded,  since  of  course  the  sputa  and  the  bowel  dis- 
charges of  all  the  tuberculous  reach  the  Elbe  thru  the  sewers;  and  tubercle 
bacilli,  just  as  well  as  typhoid  bacilli,  could  have  been  carried  thence  in 
the  water  to  the  people."  (Page  300). 

Sedgwick  and  MacNutt  find,  by  comparing  the  number  of 
deaths  from  pulmonary  tuberculosis  for  a  period  of  five  years  just 
previous  to  water-supply  purification  with  the  five-year  period  just 
following,  a  decrease  of  12  percent  at  Lawrence,  Massachusetts, 
and  a  decrease  of  14  percent  at  Lowell.  Tho  it  is  certain  that  this 
decrease  is  not  wholly  due  to  the  better  water  supply,  yet  when 
compared  with  Manchester,  Massachusetts,  a  city  with  a  fair  water 
supply,  which  supply  had  not  been  changed  during  this  ten-year 
period,  it  is  seen  that  the  percentage  of  decrease  in  deaths  from 
tuberculosis  at  Lowell  and  at  Lawrence  is  much  more  marked  than 
at  Manchester.  That  drinking  water  has  been  and  is  still,  in  places, 
a  source  of  tuberculous  infection  is  probable. 


IN   FOOD  ^e  frecluenc.v  with  which  tubercle  bacilli  occur  in 

AND  SOIL  market  milk  and  butter  is  indicated  by  a  tabular 

review  of  the  literature  upon  this  subject  given 
in  our  previous  publication  (Briscoe  and  MacNeal,  Bulletin  149 
of  this  station).  In  1233  samples  of  butter  tubercle  bacilli  were 
found  163  times  (13.2  percent);  in  7397  samples  of  market  milk 
they  were  found  502  times  (6.8  percent).  This  frequency,  to- 
gether with  the  length  of  time  that  these  germs  are  known  'to  live 
in  butter,  indicates  that  this  is  an  important  source  of  infection 
for  man,  especially  qhildren.  In  soil  and  foul  material  they  are 
known  to  live  for  a  great  length  of  time,  and  are  there  dangerous 
to  farm  animals. 

A  brief  summary  of  the  literature  on  the  life  of  tubercle  bacilli 
in  water  and  in  other  material  is  given  in  Table  7. 


1912]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  309 

TABLE  7.— DURATION  OP  L/IFE  OP  TUBERCLE  BACILT,!  IN  WATER  AND  OTHER  MATERIAI, 


Author 

Date 

Conditions 

Not  killed 

Killed 

Folk41 

1883 

In  decaying  matter  an  injury  was  ob- 
served after  a  few  days       

few  days 

Schill  and 

1884 

In  sputum,  virulent  

43  days 

Fischer129 
Sormani138 

1886 

Tuberculous  sputum  remained   viru- 
lent in  water        

12  mo. 

Chantemesse 
and  Widal22 

1888 

Sterilized    and   unsterilized    samples 
of   water    inoculated    with    pure 
cultures   of   tubercle  bacilli  were 
kept  at: 
A  temperature  of  8°  to  12°  C  

50  days 

Galtier48 

1888 

Room  temperature  15°  to  20°  C  
Tuberculous  spleen  in  water  3°  to  8°  C 
Tuberculous   products    of    pigs    and 
cows   in    running    and    stagnant 
water  at  17°  to  0°  C  

70  days 
17  days 

14  days 

» 

Tuberculous  products  of  a  cow  in  run- 
ning water  4°  to  10°  C   

2  mo. 

Cadeac  and 

1888 

Tuberculous  lung,  buried  

77  to  167 

after  176 

Malet1* 

Piece  of  tuberculous  lung  in  bowl  of 
water  exposed   on  outer  window- 
sill  

days 
76  days 

days 

Same  (repeated)     

120  day 

150  days 

Same  (repeated)  triturated  with  water, 
in  air  •  

Heim" 

1889 

In  butter  

Straus  and 
Dubarry  '  *  3 

1889 

In  river  water  (Ourcq): 
at  20°  C  

27  days 

at  35°  C  

at  38°  C  

95  days 

In  distilled  water: 
at  30°  C  

24  days 

at  35°  C     

25  days 

at  38°  C  

115  days 

Gartner44 

1890 

7  mo 

Freytag43 

1890 

Tuberculous  sputum  in  an  abundance 
of  table  salt  for  two  weeks;  then 
inoculated    under   the    skin    of  a 
guinea    pig    caused    generalized 
tuberculosis    

2  wks 

Schottelius131 

1890 

Buried  phthisical  lungs  

1891 

In  butter         

Petri113 

1891 

Organs  containing  tubercle  bacilli: 

• 

Stone140 

1891 

Ransome  and 
Delepine11  * 

1894 

Very  virulent  sputum  exposed  to  very 
little  air  in  dark  

L/oesner88 

1896 

Tuberculous  organs  placed   in   cada- 
vers of  hogs  and  buried.  ...... 

Schneiderlin 

1897 

Tubercle  bacilli  remained  8  years  in 
soil  without  losing  their  staining 
property     •  •  •  

8  vr<*  l?\ 

Gaertner48 
Hance5'- 

1898 
1898 

In  manure,  temperature  only  40°  C,  .  .  . 
Tuberculous    sputum   kept  fluid  in  a 
well'stoppered  bottle  

3^  mo. 

Klein70 

1899 

Buried  in  dead  mice  and  guinea  pigs 

7  wks. 

310 


BULLETIN  No.  161 
TABLE  7. — Continued 


[November, 


Author 

Date 

Conditions 

Not  killed 

Killed 

Musehold101 

1900 

Tuberculous  sputum  mixed  with  river 
water: 
In  diffuse  light,  room  temperature. 
In  the  dark  virulence  was  greater.  . 
Sputum  in  canal  water: 
Diff  use  light  

162  days 
162  days 

131  days 

211  days 
211  days 

197  days 

Kept  in  dark  

131  days 

197  days 

131  days 

197  days 

Sputum  in  sewage,  room  temperature 
Sputa  in  sewage: 

194  days 
194  days 

Kept  in  dark  .          

105  days 

• 

Canal   water    mixed   with    sputa  and 
garden  soil  kept  at  room  tempera- 
ture and  received  mid-day  sun.  ... 
Same    as    above    except    exposed    to 
frost    snow  and  sunshine  

145  days 
148  days 

197  days 

Same  as  above  except  kept  in  sewage 
Same  as  above  except  calcium   chlo- 
rid  added     

150  days 

Peterson111 

1900 

In  sputum  protected  from  the  sun  the 
bacilli  almost  unchanged  morpho- 

\Yz  mo. 

Patterson112 

1903 

In  butter  containing  4  percent  salt  .  . 
In  butter  containing  5  percent  salt  .  . 
In  salt  flesh    not  killed     

4  wks 

3%  wks. 
4  days 

Dixon31 
Schroeder132 

1908 
1908 

Examined  sewage  from  the  tubercu- 
lous    hospital     of     Philadelphia. 
Guinea  pigs  were  inoculated,  but 
previous   to   the   inoculation    the 
sewage   was    heated  60°  to  70°  C. 
from  2  to  IS  minutes.     No  tubercu- 
lous guinea  pigs  were  produced.  1  . 
In  butter  as  ordinarily  salted  

160  days 

Mohler,  Wash- 
burn,  Doan 

1909 

In  butter  in  cold  storage,  pure  culture 
or  tuberculous  udder  

6  mo. 

and  Rogers" 

261  days 

1  Temperature  sufficient  to  pasteurize;  most  likely  the  organisms  were  killed  by 
heating. 


EXPERIMENTAL  WORK 

The  main  purpose  of  this  work  was  to  determine 
INTRODUCTION  tne  length  of  time  that  bovine  tubercle  bacilli  live 

outside  the  animal  body  where  they  may  be  scat- 
tered by  tuberculous  farm  animals,  especially  dairy  cattle.  As  it  was 
not  feasible  to  keep  tuberculous  cattle  at  the  Station,  only  a  part  of 
the  work  originally  planned  with  naturally  infected  feces  was  done. 
In  connection  with  this  investigation  some  work  has  been  done  on 
tubercle  bacilli  of  the  human  type  and  on  other  pathogenic  an4 
non-pathogenic  organisms  for  purposes  of  comparison,- 


1912]  FATE  or  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  311 

General  Methods  of  Procedure 

There  are  three  points  that  need  special  attention:  the  sample, 
the  manner  of  exposure,  and  the  length  of  time  required  for  life 
to  become  extinct  in  the  culture  exposed. 

It  is  advisable  that  the  sample  of  the  organism 
SAMPLE  undergoing  test  be  a  pure  culture.  In  some  cases 

where  the  organism  is  readily  differentiated  from 
contaminating  growth,  this  is  not  so  essential,  as,  for  example,  when 
pathogenic  organisms  are  mixed  with  non-pathogenic  organisms 
and  the  latter  are  readily  eliminated  by  animal  inoculation,  or  when 
a  colored  organism  can  be  readily  distinguished  by  its  color  char- 
acteristic. Even  in  these  cases  it  is  usually  advisable  to  work  with 
pure  cultures. 

EXPOSURE  Much  depends  upon  the  manner  of  exposure;  for 

OF  SAMPLE  instance,  if  exposure  is  to  light  it  makes  a  decided 
difference  whether  the  culture  is  exposed  directly 
or  in  a  glass  container,  or  in  the  presence  or  in  the  absence  of 
air.  A  complete  statement  as  to  the  manner  of  exposure  is  ab- 
solutely essential  to  a  definite  interpretation  of  the  reported  re- 
sults. 


INDEX  OF  most  difficult  point  is  to  determine  when  life 

DEATH  becomes  extinct.     This    is    comparatively    simple 

with  those  organisms  that  readily  grow  on  culture 
media,  when  they  are  exposed  in  pure  culture.  Their  failure  to 
grow  gives  a  ready  index  of  their  death.  With  tubercle  bacilli  it 
it  much  more  difficult  to  tell  just  when  these  organisms  are  dead. 
They  cannot  be  cultivated  readily  from  mixed  growth  since  it  is 
very  difficult  to  prevent  over-growth  with  contaminating  organ- 
isms. Another  difficulty  is  that  dead  tubercle  bacilli  produce  typi- 
cal tuberculosis  in  test  animals  (see  page  312).  It  is  therefore 
necessary  to  make  animal  inoculations  in  all-  cases  where  mixed 
tuberculosis  materials  are  used,  and  from  the  original  test  animal, 
especially  if  only  a  local  lesion  is  produced,  a  second  healthy  ani- 
mal must  be  inoculated  from  some  of  the,  suspected  tuberculous 
material.  Suitable  culture  media  should  be  seeded  with  this 
material  from  both  the  original  and  the  secondary  test  animal. 
When  all  these  tests  prove  characteristic  for  tubercle  bacilli,  it  is 
evident  that  these  germs  are  still  living  and  virulent;  when  no  in- 
fection, or  only  a  local  lesion,  is  produced  in  either  the  original 
or  the  secondary  animal,  and  no  growth  occurs  on  the  culture 
media,  the  tubercle  bacilli  are  surely  dead.  In  cases  where  the 
tuberculosis  produced  in  the  original  inoculated  animal  from  a 
small  amount  of  material  is  severe  and  generalized,  it  may  be  con- 


312  BULLETIN  No.  161  [November. 

sidered  that  these  germs  are  living,  and  this  should  then  be  verified 
by  cultnring  on  media  suitable  for  the  growth  of  tubercle  bacilli. 

LESIONS  PRODUCED  BY  DEAD  TUBERCLE  BACILLI 
PURPOSE  OF        Tne  purpose  of  these    experiments    was  to  deter- 
MENTS  mine  the  difference  in  effect  of  dead  tubercle  bacilli 

and  of  living  germs  when  injected  into  guinea 
pigs;  also  to  find  a  means  of  determining  when  lesions  were  pro- 
duced by  living  and  when  by  dead  germs.  In  order  to  determine 
this  three  experiments  were  made. 

For  the  first  experiment  a  pure  culture  of  the 
EXPERIMENT  I  bovine  type  of  tubercle  bacilli  was  used.  A  heavy 

emulsion  of  fresh  culture  was  made  in  sterile 
distilled  water.  The  emulsion  showed  a  distinctly  milky  appear- 
ance. One  cubic  centimeter  of  this  fresh  emulsion  was  injected 
subcutaneously  into  Guinea  Pig  993.  The  remaining  portion  of 
this  emulsion  was  autoclaved  at  fifteen  pounds  pressure  for  ten 
minutes.  Two  guinea  pigs,  Nos.  994  and  995,  were  each  injected 
subcutaneously  with  I  cc.  of  the  autoclaved  emulsion.  Guinea  Pig 
993  was  killed  49  days  later  and  showed  generalized  tuberculosis. 
Guinea  Pigs  994  and  995,  killed  respectively  85  and  50  days  after 
inoculation,  were  found  to  be  healthy.  Their  weights  continued 
to  increase  from  the  time  they  were  inoculated  until  killed,  and 
at  no  time  did  they  show  any  physical  signs  of  tuberculosis. 

Before  the  conclusion  of  the  first  experiment  a 
EXPERIMENT  II  second  was  started  in  which  an  eight-day  culture 

of  the  same  strain  of  bovine  tubercle  bacilli  was 
used.  One  milligram  was  removed  and  rubbed  up  in  4  cc.  of 
broth.  One  loopful  of  this  emulsion  was  diluted  with  2  cc.  of 
broth  and  injected  interperitoneally  into  Guinea  Pig  1026.  The 
4  cc.  of  broth  were  pla,ced  in  streaming  steam  for  one  hour. 
Guinea  Pigs  1027  and  1028  were  each  injected  interperitoneally 
with  2  cc.  of  this  steamed  emulsion.  Guinea  Pig  1026  showed 
physical  signs  of  tuberculosis  ten  days  after  injection.  No  physical 
signs  of  tuberculosis  were  observed  in  either  of  the  two  guinea 
pigs  (1027  and  1028)  that  were  inoculated  with  the  killed  culture. 
Thirty-five  days  after  inoculation  Guinea  Pigs  1026  and  1027  were 
injected  with  2  cc.  of  the  tuberculin.  Guinea  Pig  1026  died  from 
the  effect  of  the  tuberculin  after  eight  hours;  1027  died  after 
eighteen  hours.  Autopsy  of  1026  showed  severe  generalized  tuber- 
culosis;  that  of  1027  showed  a  few  lesions  in  the  liver  and  a  very 
slight  enlargement  of  the  inguinal  lymphatics.  Microscopic  prep- 
arations stained  for  tubercle  bacilli  revealed  none  of  these  organ- 


1<)I2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  313 

isms.  Cultures  on  glycerine  egg  and  the  inoculation  of  a  second 
guinea  pig  gave  negative  results.  Guinea  Pig  1028  was  killed  41 
days  after  inoculation,  and  the  autopsy  showed  a  small  abcess  at 
the  point  of  inoculation  and  several  lesions  in  the  liver.  All  other 
organs  were  apparently  healthy.  A  stained  preparation  revealed 
several  tubercle  bacilli  a  part  of  which  showed  signs  of  disintegra- 
tion. Glycerine  egg  media  and  a  guinea  pig  were  inoculated  with 
pus  material  from  the  point  of  inoculation  and  a  liver  abcess. 
Neither  of  these  revealed  living  tubercle  bacilli. 

A  third  experiment  was  made  using  the  same 
EXPERIMENT  III  strain  of  tubercle  bacilli.  An  emulsion  was  made 

in  sterile  0.8  percent  salt  solution.  The  emul- 
sion showed  a  faintly  milky  appearance.  Three  tubes  of  glycerine 
egg  were  seeded  with  this  emulsion  of  tubercle  bacilli  and  Guinea 
Pig  1666  was  injected  interperitoneally  with  i  cc.  The  remaining 
portion  of  the  emulsion  was  divided  into  two  parts :  one  was 
heated  at  85°  C.  for  ten  minutes,  the  other  was  heated  in  the 
autoclave  at  115°  C.  for  ten  minutes.  With  each  of  these  portions 
three  glycerine  egg  slants  were  seeded  and  a  guinea  pig  was  in- 
oculated interperitoneally  with  I  cc.  The  unheated  portion  gave 
excellent  growth  and  produced  severe  generalized  tuberculosis  in 
Guinea  Pig  1666.  The  part  heated  to  85°  C.  for  ten  minutes  gave 
no  growth  on  either  of  the  three  glycerine  egg  tubes  after  six 
weeks  incubation  at  38°  C.  Guinea  Pig  1167,  killed  67  days  after 
inoculation,  showed  local  tuberculosis  in  the  right  superior  inguinal 
near  the  point  of  inoculation  and  a  few  small  lesions  in  the  liver. 
All  other  organs  were  apparently  healthy.  A  stained  preparation 
from  pusl  material  of  these  lesions  showed  tubercle  bacilli.  Cul- 
tures on  glycerine  egg  and  the  inoculation  of  a  second  healthy 
guinea  pig  from  this  material  gave  negative  results.  The  part 
heated  to  115°  C.  for  ten  minutes  neither  gave  growth  on  the 
glycerine  egg  media  nor  produced  any  effect  when  I  cc.  was  in- 
jected interperitoneally  into  Guinea  Pig  1168. 

i.     Dead  cultures,  when  not  killed  at  too   high  a 
CONCLUSIONS      temperature,  produce  tuberculous  lesions  in  guinea 
pigs. 

2.  Secondary  guinea  pigs  inoculated  from  tuberculous  material 
from  lesions  produced  by  dead  cultures  always  remain  healthy. 

3.  In  determining  the  length  of  time  tubercle  bacilli  live  when 
exposed  to  various  conditions  outside  the  animal  body,  it  is  nec- 
essary to  inoculate  a  second  healthy  guinea  pig,  especially    when 
only  local  lesions  are  produced  in  the  guinea  pig  inoculated  with 
the  original  material,  in  order  to  be  sure  the  tubercles  are    not 
produced  by  dead  tubercle  bacilli. 


314  BULLETIN  No.  161  [November, 

KI'I-'KCT  op  LIGHT  UPON  SPORK-  AND  NoNSPOKB-BfiARii^e 
ORGANISMS 

A  few  spore-bearing  and  nonspore-bearing  or- 
CULTURE  organisms  were  tested  by  exposing  them  to  the 

direct  rays  of  the  sun.  The  spore-bearing  organ- 
isms, B.  subtilis  and  B.  mesentericus  vulgatus,  were  grown  upon 
peptoneless  agar  (made  with  three  grams  of  beef  extract  and  fif- 
teen grams  of  agar  per  liter)  for  six  days  at  32°  C.  An  examina- 
tion of  the  cultures  showed  an  abundance  of  well-developed  spores. 
The  vegetative  cells  of  these  two  organisms  were  obtained  by  re- 
peated growths  in  beef  broth.  The  broth  cultures  were  kept  at 
37°  C.  and  repeated  every  twenty-four  hours  for  six  days.  A 
microscopic  examination  at  the  end  of  this  time  showed  no  spores. 
B.  diphtheria  cultures  were  obtained  from  twenty-four  hours' 
growth  upon  Loeffler's  blood  serum.  All  other  organisms  used  were 
from  fresh  broth  cultures  grown  at  the  optimum  temperature  for 
the  organism  tested. 


MANNER  OF         ^^e  cultlires  grown  on  the  solid  media  were  sus- 
EXPOSURE  pended  in  0.8  percent    salt    solution.     A  concen- 

tration of  slightly  milky  appearing  emulsion  was 
formed.  Then  a  loop  of  this  emulsion  was  spread  in  a  very  thin 
smear  on  either  a  small  slip  of  sterile  glazed  paper  or  a  small  sterile 
glass  cover.  These  were  then  exposed  to  the  direct  rays  of  the 
sun  for  definite  periods  of  time  between  the  hours  of  ten  in  the 
morning  and  three  in  the  afternoon.  The  exposed  slips,  with  the 
exception  of  those  containing  B.  diphtheria,  were  aseptically  drop- 
ped into  sterile  broth  and  incubated  at  a  temperature  of  30°  C.  for 
one  week.  The  exposed  smears  of  B.  diphtheria  were  seeded  on 
Loeffler's  blood  serum  and  incubated  at  37°  C.  for  one  week.  The 
cultures  were  carefully  examined  for  growth,  and  if  growth  was 
evident  further  tests  were  made  to  see  if  it  was  the  same  as  the 
original  culture  or  an  accidental  contamination. 

„__..._«  A  summary  of  the  results  is  given  in  Table  8. 

It  will  be  noted  that  the  nonspore-bearing  organ- 
isms were  all  killed  in  a  few  minutes.  (l/2  to  6 

minutes).     While  the  spore-bearing  organisms  were  not  killed  in 

the  time  exposed  (180  minutes). 


1912]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  315 

TABLE  8.— ORGANISMS  EXPOSED  TO  DIRECT  SUNLIGHT 


Name  of  organism 

Not  killed 

Killed 

B. 

120  min. 

B. 

180  min. 

B. 

mesentericus  vulgatus,  spores1  

180  min. 

B. 

subtilis,  vegetative  cells  

/^  min. 

1  min. 

B. 

mesentericus,  vegetative  cells  

1  niiii. 

2  min. 

B. 

/^  min. 

B. 

6  min. 

after  6  min. 

B. 

2  min. 

3  min. 

B. 

ty  phosus  

1  min. 

2  min. 

B. 

violaceus  

2  min. 

3  min  . 

'Time  of  exposure  was  not  continued  longer;  no  end  point  was  reached. 


EFFECT  OF  DIRECT  SUNLIGHT  UPON  TUBERCLE  BACILLI 


CULTURE  AND  f  direct  sunlight  was    tested    several 

EMULSION  times  upon  cultures  from  three  types  of  tubercle 

bacilli.  The  organisms  used  for  exposure  were 
always  from  active  cultures  grown  upon  glycerine  egg  from  two  to 
three  weeks.  A  heavy  emulsion  was  made  by  rubbing  up  some  of 
the  culture  on  the  inside  of  the  neck  of  a  sterile  glass-stoppered 
graduate  flask  with  a  sterile  glass  rod.  From  time  to  time  a  few 
drops  of  sterile  0.8  percent  salt  solution  were  added.  At  first 
the  culture  was  rubbed  into  a  fine  paste  with  the  addition  of  only 
a  few  drops  of  the  salt  solution  ;  then  about  6  cc.  of  0.8  percent 
salt  solution  were  added  and  the  contents  shaken  thoroly.  The 
emulsion  then  showed  a  decidedly  milky  appearance  and  was 
filtered  thru  sterile  glass  wool  to  remove  the  larger  clumps.  The 
examination  of  a  stained  preparation,  made  in  a  similar  way  as 
the  smears  that  were  used  for  exposure  to  sunlight,  showed  the  in- 
dividual organisms  usually  well  separated.  There  were,  however, 
clumps  of  twenty-five  to  thirty  organisms  still  to  be  found. 

PREPARATION     Smears  from  the  prepared  emulsions  were  made 
AND  EXPOSURE  UpOn  small  slips  of    sterile    glazed    paper.     Pins 

OF  SMEARS  F  i      ,    .,          ,,F  r  ,,&  .. 

pushed  thru  the  corners  of  these  small  paper  slips 
were  then  stuck  into  the  bottom  of  a  pasteboard  box  having  a  snug 
fitting  lid.  The  box  and  the  slips  were  sterilized  at  150°  C.  for 
one  hour.  A  small  loop  f  til  of  the  emulsion  was  smeared  in  a  thin 
layer  upon  each,  of  the  sterile  glazed  paper  slips.  These  were  ex- 
posed at  once  to  the  sun  for  the  desired  length  of  time.  Triplicate 
smeared  slips  for  each  period  of  exposure  were  seeded  upon  culture 
media  suitable  for  the  growth  of  tubercle  bacilli.  The  media  iised 
was  5  percent  glycerine  beef-juice  agar  or  glycerine  egg. 


316 


BULLETIN  No.  161 


[November, 


The  glycerine  egg  was  prepared  by  opening,  aseptically,  ten 
good  fresh  eggs  and  pouring  the  whole  contents  into  a  sterile  flask. 
To  the  well-beaten  eggs  there  were  added  200  cc.  of  5  percent  gly- 
cerine beef  broth.  This  emulsion  was  placed  in  sterile  test  tubes 
by  means  of  a  sterile  Pasteur's  bulb  pipette.  It  was  solidified,  in 
a  slanting  position,  at  73°  C.  for  two  hours.  After  placing  the 
exposed  slip  on  the  surface  of  the  media,  it  was  rubbed  and  scraped 
by  means  of  a  small  sterile  platinum  spatula  so  as  to  dislodge  some 
of  the  dried  tubercle  bacilli,  and  a  small  amount  of  the  glycerine 
egg  or  glycerine  agar  was  smeared  over  this  slip  until  it  was 
thoroly  moistened.  The  cotton  stoppers  were  flamed,  dipped  into 
paraffine  and  pushed  into  the  tubes,  which  were  then  stopped  with 
paraffined  stoppers.  The  cultures  were  incubated  at  38°  C.  for  one 
month  and  then  carefully  examined  for  growth. 

Table  9  indicates  the  method  of  recording  the  re- 
RESULTS  suits  for  the  separate  tests.  A  summary  of  the 

results  of  the  various  tests  from  the  three  types 
of  cultures  is  given  in  Table  10.  It  is  seen  that  these  germs  are 
killed  in  a  very  short  time  (i  to  4  minutes).  These  results  agree 
with  those  of  Weinzirl  (see  Table  2). 


TABLE  9.— BACILLUS  TUBERCULOSIS,  HUMAN  TYPE  EXPOSED  TO  SUNSHINE 
(Between  11  and  12  A.  M.,  Nov.  2,  1911) 


Time  of 
exposure, 
minutes 

Conditions 

Result  of 
growth 

Remarks 

0 
0 
0 
% 

% 

Y* 

i 
i 

2 
2 
2 
4 
4 
4 
6 
6 
6 
10 
10 
10 

C< 

S 

V 

jntrols 
« 

II 

in  bri 

w  clc 

i 

< 

5,  not 
(i 

Fht>  n 
uds  n 

sxpos 

<  i 

« 
o  cloi 

• 

ar  tl 

ed  

+ 

+ 
+ 

f? 

+ 

Excelh 

« 

it 
ii 

Contarr 
Excelle 
Modera 

No  gro 
Con  tat 

:nt  growth 
« 

« 

<  i 
« 

ination 
nt  growth 

te  growth 
«           • 

wth 
ination  mould 

ads  

? 

le  horizon 

i         « 

<         <i 

i<)i2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  317 

TABLE  10 — SUMMARY  ot?  THE  RESULTS, FROM  EXPOSING  TUBERCLE  BACILLI 

TO  DIRECT  SUNSHINE 


Name  of  culture 

Not  killed, 
minutes 

Killed, 
minutes 

B. 

tuberculosis,  human  

1 

2 

B. 

1 

2 

B. 

tuberculosis,  avian  

2 

4 

OF  DESICCATION  UPON  BACTERIA 

PURPOSE  ^le  PurP°ses  °f  this  work  was  to  compare  theef- 

OF  WORK  feet  of  desiccation  upon  tubercle  bacilli  with  that 

upon  some  nonacid-fast  bacteria.  Two  types  of 
tubercle  bacilli  were  used,  human  and  bovine.  The  nonacid-fast 
organisms  used  were  two  pathogenic  organisms,  B.  typhosus  and 
B.  diphtheria,  and  of  the  nonpathogenic  germs  sporebearing  and 
nonspore-bearing  were  used  (see  list  in  Table  n). 


CULTURES  AND  ^e  cultures  were  grown  and  the  emulsions  made 
EMULSIONS  in  the  same  way  as  described  in  the  section  on  the 
effect  of  light  upon  bacteria.  Only  fresh,  active 
cultures  were  used.  The  tubercle  bacilli  were  grown  only  upon 
glycerine  egg. 

MANNER  OF        ^he  manner  of  preparing  the  organisms  for  ex- 
EXPOSURE  posure  was  also  the  same  as  that  described  in  the 

last  section,  with  the  exception  that  the  smears 
were  made  only  upon  sterile  glazed  paper  slips.  These  smeared 
slips  were  exposed  in  a  dark  sheet-iron  box  that  was  well  ventilated. 
Control  slips  of  all  the  organisms  tested  were  seeded  a  few  minutes 
after  the  visible  moisture  had  disappeared  from  the  paper  slips. 
Thereafter  duplicate  slips  were  seeded  at  the  end  of  12  hours  and 
i,  2,  4,  8,  12,  and  16  days.  The  tubercle  bacilli  were  cultivated 
upon  glycerine  egg  at  38°  C.  for  six  weeks.  In  examining  the  cul- 
tures for  growth  of  these  germs  it  is  necessary  to  scrape  the  surface 
and  make  stained  preparations  even  when  visible  growth  is  not 
present,  for  in  practically  every  case  there  will  be  one  or  two  cul- 
tures between  the  evidently  visible  growth  and  the  culture  proven 
to  contain  no  growth  by  the'  absence  of  organisms  in  stained  pre- 
parations, in  which  the  cultures  will  have  only  a  few,  usually  in- 
visible, colonies.  This  condition  was  not  noted  in  the  cultures 
from  smears  exposed  to  the  sun. 

The  smears  of  B.  diphtheria  were  cultivated  upon  Loeffler's 
blood  serum  at  37°  C.,  and  those  of  all  other  organisms  were  placed 
in  broth  and  cultivated  at  the  optimum  temperature  for  each  species. 


318 


BULLETIN  No.  161 


[November 


RESULTS 


The  results  are  given  in  Table  i  T  .  These  are  the 
average  results  from  two  testings  of  all  organisms 
used.  Some  other  tests  were  made,  but  the  re- 
sults are  only  slightly  different  from  the  average  of  these  two 
complete  series.  In  each  of  these  two  series  the  organisms  were 
all  tested  at  the  same  time  and  under  the  same  conditions,  with 
the  exception  that  the  cultures  were  grown  upon  different  media, 
as  noted  above.  This  may  have  made  some  difference  in  the 
results.  The  tubercle  bacilli  were  grown  upon  glycerine  egg  media 
for  approximately  three  weeks,  while  B.  violaceus,  for  instance, 
was  grown  in  broth  for  24  hours  at  30^  C.  The  tubercle  bacilli 
were  emulsified  in  0.8  percent  salt  solution  and  transferred  to  the 
sterile  paper  slips,  while  the  B.  liolaceus  were  transferred  directly 
from  the  broth  to  the  slips.  It  may  be  that  at  least  part  of  the 
difference  in  the  length  of  time  required  to  kill  these  organisms 
by  desiccation  is  due  to  this  difference  of  growth  and  age  of  the 
cultures. 

It  is  seen  that  a  very  little  longer  time  is  required  to  kill  the 
tubercle  bacilli  than  other  nonspore-bearing  organism;  and  this 
slight  difference  may  readily  be  produced  by  the  protection  of  the 
glycerine  clinging  to  these  organisms  from  the  culture  media,  or 
more  likely  the  protection  offered  by  the  presence  of  clumps  of 
tubercle  bacilli.  It  is  almost  impossible  to  eliminate  clumps  from 
an  emulsion  of  tubercle  bacilli,  while  other  organisms  repeatedly 
grown  in  broth  may  be  well  separated. 

The  tubercle  bacilli,  therefore,  cannot  be  put  in  the  class  of 
bacteria  with  spores,  and  they  are  very  near  to  nonspore-bearing 
organisms,  or  the  vegetative  cells  of  spore-bearings  organisms,  as 
regards  the  effect  of  desiccation.  They  should  be  classed  with  the 
more  resistant  of  the  nonspore-bearers. 

TABLE  11.— EFFECT  OF  DESICCATION  UPON  BACTERIA 


Organism 

Not  killed, 
days 

Killed, 
days 

B. 

tuberculosis,  human  

4 

8 

B. 

tuberculosis,  bovine  

8 

12 

B. 

diphtheria  

4 

5 

B. 

typhosus  

3 

5 

B 

subtilis,  spores  

35 

B. 

subtilis,  vegetative  cells  

1 

2 

B. 

35 

B. 

vulgatus,  vegetative  cells  

2 

3 

B. 

coli  

3 

4 

B. 

violaceus  

1 

2 

B. 

prodi°"iosus  

1 

9 

Sar 

lutea  

4 

5 

Sar. 

4 

5 

I9i-?]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  319 

DURATION  of  LIFE)  OF  BOVINE  TUBERCLE  BACILLI  IN  Cow 

MANURE 

PURPOSES  OF      Tne  general  purposes  of  these  experiments  was  to 
MENTS  determine  how  long  bovine    tubercle    bacilli    will 

live  in  cow  manure  when  exposed  to  weather  con- 
ditions in  a  pasture  field.  Two  series  of  tests  have  been  made  con- 
cerning the  resistance  of  these  organisms  when  a!  pure  culture  is 
artificially  mixed  in  cow  manure  exposed  in  the  sunshine,  and  also 
when  it  is  exposed  in  a  place  protected  from  the  sunshine.  In  a 
similar  way  other  tests  have  been  made  with  naturally  infected 
manure  from  a  tuberculous  cow. 

Series  i 
(Special  Methods) 

PREPARATION  Artificially  Infected  Manure. — The  culture  used 
OF  SAMPLES  in  preparing  this  sample  of  artificially  infected 
manure  was  a  strain  of  bovine  tubercle  bacilli  ob- 
tained from  Dr.  Theobald  Smith  of  the  Harvard  Medical  School. 
It  was  received  and  has  since  been  kept  upon  glycerine  agar.  The 
strain  readily  produces  an  abundant  growth  and  is  quite  virulent 
to  test  animals.  * 

Four  milligrams  of  the  culture  from  two  tubes  of  glycerine 
agar  were  removed  and  emulsified  as  described  on  page  315.  The 
emulsion  was  diluted  to  200  cc.  with  sterile  salt  solution.  It  was 
expected  that  the  large  amount  of  solution  used  would  give  a 
more  uniform  mixture  of  the  tubercle  bacilli  in  the  cow  manure. 

The  sample  of  manure  was  obtained  from  two  cows  of  the 
dairy  herd.  It  was  taken  by  the  method  described  in  Bulletin  149 
of  this  station;  i.  e.,  by  injecting  air  into  the  rectum  of  the  cow 
until  stimulated  to  defecate.  The  feces  were  caught  in  a  sterile 
pail  and  at  once  covered  and  brought  to  the  laboratory.  The  200 
cc.  emulsion  of  bovine  tubercle  bacilli  was  thoroly  mixed  with 
1800  grams  of  the  fresh  cow  manure.  The  infected  manure  was 
tested  for  virulence  by  inoculation  of  two  guinea  pigs.  One  gram 
was  rubbed  up  in  50  cc.  of  0.8  percent  salt  solution,  40  cc.  of  the 
emulsion  was  centrifuged  and  the  sediment  injected  subcutaneously 
into  two  guinea  pigs,  both  of  which  became  infected  with  gen- 
eralized tuberculosis. 

Naturally  Infected  Manure. — The  sample  of  naturally  infected 
manure  was  obtained  from  a  tuberculous  cow  (cow  No.  56  from 
the  dairy  herd  of  this  station)  that  had  previously  reacted  to 
tuberculin.  The  sample  was  taken  by  the  method  described  in  our 
previous  publication  referred  to  above.  Approximately  three  kilos 


320  BULLETIN  No.  161  [November, 

of  fresh  manure  were  obtained.  The  manure  from  this  cow  had 
been  tested  a  number  of  times  for  tubercle  bacilli  by  making  sub- 
cutaneous inoculations  of  I  cc.  of  a  2  percent  emulsion  of  the 
fresh  f  eces.  Upon  four  occasions  such  tests  gave  negative  results  ; 
three  other  tests,  made  respectively  August  31,  1909,  July  18, 
1910,  and  August  16,  1910,  produced  tuberculous  guinea  pigs. 
The  guinea  pigs  in  none  of  the  three  tests  became  severely  tuber- 
culous ;  and  two  of  the  guinea  pigs,  one  each  in  the  last  two  tests, 
remained  healthy.  The  last  testing,  August  16,  1910,  was  made 
on  the  day  of  exposing  this  sample  of  manure  to  the  weather. 
Tho  the  infected  guinea  pig  from  this  sample  did  not  show  severe 
tuberculosis  when  killed  and  examined  80  days  after  inoculation, 
a  second  guinea  pig  inoculated  with  the  diseased  tissue  from  this 
guinea  pig  showed  severe  generalized  tuberculosis  when  killed  and 
examined  38  days  later. 


EXPOSURE  OF  infected  manures  were  taken  to    a    secluded 

SAMPLES  plot  of  ground  on  the  Experiment  Station  farm 

for  exposure.  The  manure  infected  with  pure 
culture  of  bovine  tubercle  bacilli  was  divided  into  two  equal-  parts. 
One  half  was  placed  in  a  free,  open  place,  fully  exposed  to  the 
sunshine  thruout  the  whole  day.  This  part,  flattened  out  into  a  two- 
inoh  layer,  was  placed  upon  a  sod  with  the  grass  cut  short.  A 
one-inch  mesh  poultry  netting  was  placed  over  the  infected  manure 
in  order,  especially,  to  keep  out  the  English  sparrows,  so  that 
they  could  not  carry  upon  their  feet  this  infected  manure  to  the 
stock  upon  the  Experiment  Station  Farm. 

The  other  part,  protected  from  the  sunshine,  was  placed  a  few 
yards  from  the  former  on  the  north  side  and  very  near  a  bank  of 
earth  six  feet  high.  The  ground  upon  which  this  manure  was 
exposed  was  first  made  smooth  by  removing  the  sod.  The  manure 
was  then  spread  in  a  two-inch  layer  just  as  was  the  part  exposed  in 
the  sunshine.  To  protect  this  sample  further  from  the  light,  an  em- 
bankment of  soil  was  made  one  foot  high  on  three  sides,  and  was 
covered  over  with  a  bunch  of  weeds.  Tho  the  soil  was  more 
moist  than  in  the  plot  where  the  part  was  exposed  in  the  sunshine, 
it  was  not  more  moist  than  that  usually  found  in  a  shady  place.  The 
layer  of  infected  manure  in  this  protected  place  dried  on  top  in 
a  week's  time  to  a  hard  crust,  but  trie  bottom  always  remained 
moist.  The  manure  from  the  tuberculous  cow  was  divided  into 
two  parts;  one  part  was  placed  in  the  sunshine,  and  the  other  in 
the  place  protected  from  the  sunshine,  very  near  the  two  artificially 
infected  samples  and  exposed  in  the  same  way. 


iyi2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  321 

TCCTIM^  -rue      The  samples  were  tested    almost    exclusively    by 

Tea  I  IINla    I  nt         _  .  .  .  .,  J  ,      ', 

SAMPLES  inoculation  of  guinea  pigs  following  the  method 

outlined  in  our  previous  publication.  Stains  of 
smears  from  the  infected  manure  were  made  on  a  few  occasions; 
but  since  it  is  impossible  to  distinguish  between  dead  and  living 
tubercle  bacilli  by  examining  stained  preparations,  no  reliance  is 
placed  upon  this  test.  The  largest  sample  possible  was  used  so 
as  not  to  kill  the  guinea  pigs  by  acute  infection.  The  amount  of 
sample  varied,  using  the  centrifuge  sediment  from  40  cc.  of  a  1^2 
to  a  2^2  percent  emulsion  of  dried  feces  in  0.8  percent  salt  solu- 
tion. The  sample  from  the  layers  of  infected  manures  was  taken 
by  cutting  out  pieces  of  the  dried  layer  about  one  inch  square  in 
cross  section  and  taking  all  the  manure  in  this  section  down  to 
the  ground  and  a  small  layer  of  soil  with  the  sample,  so  as  to  be 
sure  to  get  any  tubercle  bacilli  that  might  have  passed  into  this 
top  layer  of  soil  just  under  the  infected  manure. 

The  testing  of  the  sample  was  carried  out  with  more  than 
usual  precautions,  keeping  in  mind  all  the  time  the  probability  of 
producing  tubercles  in  the  guinea  pig,  with  dead  tubercle  bacilli. 
To  be  sure  that  the  tubercles  in  the  infected  guinea  pigs  were 
produced  by  living,  virulent  bacilli,  the  extent  and  rapidity  of  the 
disease  was  considered,  and  also  the  diseased  tissues  were  tested 
for  tubercle  bacilli  by  staining  smears,  by  cultures,  and  by  inocula- 
tion of  another  guinea  pig.  If  the  second  guinea  pig  became 
tuberculous,  stains  and  cultures  for  tubercle  bacilli  from  its  dis- 
eased tissues  were  made.  If,  now,  all  these  tests  were  character- 
istic for  tubercle  bacilli,  it  was  considered  reasonably  certain  that 
the  tubercle  bacilli  found  in  these  diseased  tissues  were  alive  and 
virulent.  But  when  the  guinea  pig  inoculated  from  the  sample  of 
manure  became  in  a  short  time  severely  tuberculous,  it  was  not 
thought  necessary  always  to  inoculate  a  second  guinea  pig  from 
the  diseased  tissues  of  the  former. 


WEATHER  weatner  conditions  during  the  time  the  ma- 

CONDITIONS  nure  infected  with  tubercle  bacilli  was  exposed  are 
given  in  Table  I2.1  The  weather  was  not  marked 
with  any  unusual  occurrences.  With  the  exception  of  the  loss  of 
the  sunshine  records  from  the  third  to  the  seventh  of  September, 
inclusive,  when  the  electric  sunshine  recorder  failed  to  work,  the 
records  are  quite  complete.  This  table  records  the  date  on  which 
each  test  was  made,  the  number  of  days  of  exposure  to  the  time 


1The  data  for  this  table  were  furnished  us  from  the  L/aboratory  of  Soil 
Physics  of  the  Department  of  Agronomy  of  this  station  by  the  kindness  of 
Professor  J.  G.  Hosier. 


322 


BULLETIN  No.  161 


[November, 


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I<)i2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  323 

of  the  test;  the  number  of  clear  days  during  this  time  and  also  the 
number  of  partly  and  of  wholly  cloudy  days.  There  is  also  given 
the  number  of  hours  of  sunshine,  the  amount  of  rainfall,  the 
highest  maximum  daily  temperature  and  the  lowest  minimum  daily 
temperature  occurring  between  each  test;  and  an  average  daily 
maximum,  and  an  average  daily  minimum  temperature  during  the 
time  of  exposure  up  to  the  time  the  test  was  made.  There  is 
also  given  the  average  monthly  temperature  occurring  during  the 
time  of  exposure. 

RESULTS  OP  THE  TESTS 

FROM  ARTIFI-  Exposed  in  the  Sunshine. — The  results  of  testing 
FECTED  t^ie  samples  °f  cow  manure  infected  with  a  pure 

MANURE  culture  of  tubercle  bacilli  exposed  in  the  sunshine 

are  given  in  Table  13.  As  previously  mentioned 
(page  320),  the  test  of  the  sample  on  July  29,  1910,  the  day  it  was 
first  exposed,  produced  two  severely  tuberculous  guinea  pigs.  Since 
exposure  seven  tests  have  been  made.  On  the  7th  and  i6th  days 
the  four  guinea  pigs  inoculated  contracted  severe  generalized  tuber- 
culosis. Only  miscroscopic  and  cultural  tests  were  made  from  the 
two  guinea  pigs  inoculated  on  the  /th  day  of  exposure,  since  the 
infection  in  both  guinea  pigs  was  so  severe  it  appeared  certain  to 
be  from  living  tubercle  bacilli.  This  was  later  shown  to  be  true 
from  the  culture  tests.  Microscopic  and  culture  tests  from  the 
diseased  tissues  of  both  of  the  tuberculous  guinea  pigs  infected 
with  the  sample  taken  on  the  i6th  day  of  exposure,  as  well  as  an 
inoculation  test  from  the  tissues  of  one  of  them,  showed  charac- 
teristic tubercle  bacilli.  The  test  of  the  sample  made  31  days  since 
first  exposed  produced  in  both  guinea  pigs  inoculated  only  slight 
tuberculosis  of  the  right  superior  inguinal  lymphatics.  Microscopic, 
cultural,  and  inoculation  tests  of  the  diseased  tissues  from  these 
guinea  pigs  showed  typical  tubercle  bacilli.  On  the  49th  day  of 
exposure  (September  16,  1910)  the  test  showed  that  the  virulence 
of  the  tubercle  bacilli  had  considerably  decreased,  but  was  sufficient 
to  produce  slight  tuberculosis  in  one  of  the  two  guinea  pigs  in- 
oculated. The  guinea  pigs  Were  inoculated  subcutaneously  with 
the  centrifuge  sediment  of  40  cc.  of  an  emulsion  made  by  thoroly 
grinding  three  grams  of  the  dried  sample  of  infected  manure  in 
150  cc.  of  0.8  percent  salt  solution.  The  pus  from  the  right  supe- 
rior inguinal  of  the  guinea  pig  that  became  tuberculous  was  shown 
by  miscroscopic,  cultural,  and  inoculation  tests  to  contain  charac- 
teristic tubercle  bacilli.  The  other  guinea  pig,  when  killed  53 
days  after  inoculation,  was  found  to  be  healthy.  Tests  made  on 


324 


BULLETIN  No.  161 


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326  BULLETIN  No.  161  [November, 

the  8 1st,  1 23d  and  I7ist  days  of  the  exposure  to  sunshine  showed 
the  tubercle  bacilli  to  be  dead  or  at  least  not  sufficiently  virulent  to 
produce  tuberculosis  when  the  centrifuge  sediment  was  injected  sub- 
cutaneously  into  guinea  pigs. 

It  thus  appears  from  these  tests  that  pure  cultures  of  bovine 
tubercle  bacilli,  when  mixed  with  cow  manure  and  exposed  in  an 
open  place  in  a  pasture  field,  remained  alive  in  this  instance  for  ap- 
proximately two  months. 

Exposed  in  the  Shade. — The  part  of  the  artificially  infected 
manure  exposed  in  a  place  protected  from  sunshine  was  tested  on 
the  same  days  and  in  the  same  manner  as  was  the  part  exposed 
in  the  sunshine.  These  results  are  given  in  Table  14.  The  parts 
tested  on  the  7th,  i6th,  3ist  and  49th  days  were  shown  at  each 
testing  to  contain  virulent  tubercle  bacilli.  The  infection  of  the 
guinea  pigs  in  the  first  two  tests  was  severe.  The  guinea  pigs  in- 
oculated with  samples  taken  on  the  3ist  and  49th  days  of  the 
exposure  were  infected  with  tuberculosis  but  not  so  severely  as 
the  guinea  pigs  inoculated  on  the  7th  and  i6th  days.  However, 
they  were  much  more  severely  infected  than  the  guinea  pigs  in- 
oculated with  the  sample  taken  from  the  part  exposed  in  the  sun- 
shine and  tested  upon  the  same  days.  The  three  samples  taken 
on  the  8ist,  I23d  and  I7ist  days  were  shown  not  to  contain  viru- 
lent tubercle  bacilli.  Three  of  the  guinea  pigs  inoculated  with  the 
first  two  samples  remained  healthy  until  killed  on  the  49th  and 
55th  days  respectively  after  inoculation  of  the  sample.  One  of 
the  two  guinea  pigs  inoculated  with  the  sample  taken  on  the  8ist 
day  died  five  days  later  with  an  acute  infection.  The  two  guinea 
pigs  inoculated  with  the  sample  taken  on  the  I7ist  day  died  of 
acute  infection  with  no  evidence  of  tuberculosis. 

From  these  tests  it  appears  that  tubercle  bacilli  mixed  with 
cow  manure  remain  virulent  to  guinea  pigs  for  49  days  after  ex- 
posure in  a  place  protected  from  the  sun.  All  tests  made  later 
than  this  date  showed  these  organisms  to  have  lost  their  virulence. 
Virulence  was  retained  longer  in  the  shade  than  in  the  sunshine, 
as  shown  by  the  production  of  more  severe  tuberculosis  in  the 
guinea  pigs  inoculated  from  samples  exposed  in  the  shade  than  that 
produced  in  the  guinea  pigs  inoculated  on  the  same  days  with  sam- 
ples exposed  in  the  sunshine. 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  327 

FROM  NATUR-      The  tests  of  the  samples  of  manure    from    the 
ALLY  INFECTED  tuberculous  cow,  both  the  one    exposed    in    the 

MANURE  ,       ,  ,     .  j   •      xi  i  • 

shade  and  the  one  exposed  in  the  sunshine,  at  no 
time  after  exposure  produced  tuberculous  guinea  pigs.  The  re- 
sults are  tabulated  in  Tables  15  and  16.  The  sample  exposed  in 
the  sunshine  was  tested  on  the  I3th,  34th,  63d,  and  io5th  days 
after  first  exposure.  All  the  guinea  pigs  save  one,  which  died  of 
acute  infection  in  four  days,  remained  healthy  until  killed  and 
examined  49  and  52  days  after  inoculation.  The  sample  exposed 
in  the  place  protected  from  sunlight  was  tested  on  the  same  days 
as  the  one  exposed  in  sunshine,  with  one  exception.  No  test  was 
made  on  the  I3th  day  of  exposure  from  this  part  of  the  infected 
manure  since  there  were  not  a  sufficient  number  of  guinea  pigs 
available  at  that  time.  This  sample  was  omitted  because  it  was 
thought  that  the  tubercle  bacilli  in  the  part  protected  from  the 
sun  would  be  the  least  likely  to  die.  It  was  indeed  unexpected  that 
these  bacilli  would  be  dead  in  either  of  these  two  sampled  at  this 
time.  It  was  a  hot  time  in  August.  During  the  exposure  from 
August  1 6  to  29  there  was  an  average  temperature  of  72.32°  F. 
and  a  rainfall  of  2.32  inches,  having  six  clear,  one  wholly  cloudy, 
and  seven  partly  cloudy  days.  A  number  of  showers  occurred, 
making  it  an  excellent  time  for  the  growth  of  decay  organisms 
found  in  the  manure. 

The  killing  of  the  tubercle  bacilli  in  so  short  a  time  was  no 
doubt  due  partly  to  the  antagonism  of  the  decay  organisms  and 
partly  to  the  weakened  virulence  of  these  germs.  Slight  virulence 
was  shown  by  the  producing  of  only  localized  tuberculosis  in  one 
of  the  two  control  guinea  pigs  inoculated  with  a  sample  of  the 
fresh  manure.  More  experimental  data  upon  this  subject  is  very 
desirable. 


328 


BULLETIN  No.  161 


[November, 


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330  BULLETIN  No.  161  >  [November, 

Series  2 

PREPARATION     A  second  series  of  experiments  exposing  cow  ma- 
AND  EXPOSURE  nltre  artificially  infected  with  a  pure  culture    of 

OF  SAMPLES          ,        .         ,    «         ,      t       •„.  ATA, 

bovine  tubercle  bacilli  was  made,  ine  prepara- 
tion and  exposure  of  these  samples  were  made  in  the  same  man- 
ner as  in  Series  I  (see  pages  319  and  320).  The  places  of  exposure 
were  within  a  few  feet  from  the  places  where  the  samples  were  ex- 
posed in  the  first  series. 

WEATHER  Table  17  gives  the  weather  conditions  during  the 

CONDITIONS  time  of  exposure  of  this  series.  The  same  plan 
is  followed  as  in  Table  12  giving  the  weather 
conditions  in  the  first  series.  These  conditions  were  just  the  re- 
verse of  those  in  the  first  series,  the  first  series  beginning  in  the 
hot  month  of  August,  the  second  in  the  cooler  and  more  moist 
month  of  March.  No  especially  unusual  weather  conditions  oc- 
curred during  this  period. 

Manure  'Exposed  in  the  Sunshine. — The  results  of 
RESULTS  exposing  this  infected  manure  in  the  sunshine  are 

given  in  Table  18.  The  tubercle  bacilli  were  still 
alive  at  the  third  test  after  45  days  of  exposure.  Tho  the  tuber- 
culosis produced  in  each  of  the  two  guinea  pigs  from  this  test  was 
only  slight,  as  shown  when  these  guinea  pigs  were  killed  and  ex- 
amined 53  days  later,  Guinea  Pig  973,  inoculated  with  the  dis- 
eased tissues  from  these  two,  produced  severe  generalized  tuber- 
culosis. Typical  cultures  were  obtained  from  the  diseased  tissues 
of  both  the  original  and  the  secondary  inoculated  guinea  pigs. 
The  samples  taken  after  this  date  produced  no  tuberculosis  in  the 
six  guinea  pigs  inoculated. 

Manure  Exposed  in  the  Shade. — The  results  of  exposing  the 
sample  in  the  shade  are  given  in  Table  19.  Here  we  find  that  the 
tubercle  bacilli  were  alive  for  73  days,  while  in  the  sample  ex- 
posed in  the  sun  they  were  dead  at  this  testing.  Also  the  test 
made  on  the  45th  day  shows  more  severe  tuberculosis  in  the  guinea 
pigs  inoculated  from  the  sample  kept  in  the  shade  than  from  the 
sample  in  the  sun. 

i.  As  shown  by  the  results  in  both  series  of  ex- 
CONCLUSIONS  periments,  a  pure  culture  of  bovine  tubercle  bacilli 

mixed  with  cow  manure  and  exposed  in  the  sun- 
shine in  a  pasture,  remains  alive  and  virulent  for  approximately 
two  months. 

2.  The  virulence  of  the  tubercle  bacilli  in  cow  manure  was  re- 
tained in  the  samples  protected  from  the  sunshine  longer  than  in 
those  exposed  in  the  sun,  as  shown  both  by  the  increased  length 


1912}  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


331 


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FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


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334  BULLETIN  No.  161  [November, 

of  time  that  they  remained  alive  and  by  the  severity  of  the  disease 
produced  in  the  guinea  pigs  inoculated  upon  the  same  days  from 
each  of  these  samples.  This  difference,  however,  was  doubtless 
due  mainly  to  the  difference  in  drying. 

3.  Tubercle  bacilli  in  the  manure  of  a  naturally  infected  cow 
were  dead  within  two  weeks  after  exposure.  More  experimental  re- 
sults are  necessary  before  trustworthy  conclusions  can  be  drawn. 

4.  Danger  of  infecting  farm  animals  from  tuberculous  ma- 
nure is  indicated. 

i 
DURATION  OF  LIFE  OF  BOVINE  BACILLI  IN  GARDEN  SOIL 

DIIDDOCC  Some  investigators  state  that  tubercle  bacilli  will 

KUKrUot  .,,.,,  ,         .      ,  , 

OF  TEST  live  in  soil  and  in  dead  carcasses  buried    in    the 

soil  from  two  to  three  years.  If  this  be  true,  ma- 
nure from  tuberculous  cattle  when  put  upon  cultivated  fields  which 
are  later  used  to  pasture  stock  may  be  a  source  of  infection  for  farm 
animals,  especially  hogs.  For  this  and  other  reasons  it  was  thought 
advisable  to  test  the  length  of  time  a  pure  culture  of  bovine  tubercle 
bacilli  will  live  in  garden  soil. 

The  culture  of  bovine  tubercle  bacilli  was  of  the 
CULTURE  same  strain  as  that  used  in  the  experiment  forarti- 

fically  infecting  the  cow  manure.  About  four  milli- 
grams of  pure  culture  were  obtained  from  a  four-weeks'  growth  on 
the  surface  of  two  large  tubes  of  glycerine  agar.  The  organisms 
were  carefully  removed  from  the  glycerine  agar  and  emulsified  as 
described  under  "Cultures  and  Emulsions,"  page  315. 

CA..DI  c  The  sample  of  soil  with  which  the  tubercle  bacilli 

oAl¥ir*LC  .  .  ...... 

OF  SOIL  emulsion  was  mixed  was  obtained  from  a  garden 

plot  that  had  been  in  cultivation  only  two  years. 
Previous  to  that  time,  this  plot  of  ground  had  been  in  sod  for  at 
least  fifteen  years.  The  part  from  which  the  soil  sample  was  ob- 
tained had  been  well  manured  with  horse  manure  the  first  year  it 
was  under  cultivation.  The  second  year  no  manure  was  added  but 
the  ground  was  well  stirred  and  made  into  a  lettuce  bed.  A  suf- 
ficient amount  of  this  soil  was  obtained  to  fill  a  ^4 -inch  mesh  wire 
basket  having  the  dimensions  of  4x5x6'  inches.  This  required 
1700  grams.  This  pulverized  soil  was  placed  in  a  large  pan  and 
the  emulsion  of  tubercle  bacilli  sprinkled  over  it.  These  were 
thoroly  mixed  by  being  constantly  stirred  for  some  time.  The 
amount  of  emulsion  was  sufficient  to  make  the  soil  quite  wet  and 
stickv. 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  335 


PLACE  OF  grams  ot  this  infected  soil  were  removed  to 

EXPOSURE  be  tested  for  tubercle  bacilli  as  a  control.    The  re- 

maining soil  was  put  into  the  wire  basket  and 
covered  with  a  wire  gauze.  It  was  buried  in  another  garden  that 
had  been  under  cultivation  for  one  year  and  had  received  no  ma- 
nure or  fertilizer  of  any  kind.  The  place  where  it  was  buried  had 
been  previously  well  pulverized.  The  basket  with  the  infected  soil 
was  buried  six  inches  under  the  surface  of  the  ground. 

TESTS  OF  Samples  of  this  infected  soil  were  tested  for  tuber- 

SAMPLES  cle  bacilli  on  the  first  day  of  exposure  and  on  the 

7th,  1  6th,  34th,  55th,  and  thereafter  about  once 
a  month  for  352  days.  Ten  grams  of  soil  were  removed  by  dig- 
ging down  beside  the  mouth  of  the  basket  and  with  a  sterile  pota- 
to knife  making  an  opening  to  the  center  of  the  basket.  After  the 
removal  of  the  sample,  the  opening  was  filled  by  pressing.  the  soil 
in  around  this  opening  with  a  potato  knife.  The  wire  gauze  was 
placed  over  the  mouth  of  the  basket  and  the  garden  earth  filled  in 
over  it  to  a  depth  of  six  inches.  The  soil  sample  taken  to  the  lab- 
oratory was  thoroly  shaken  with  200  cc.  of  0.8  percent  salt  solu- 
tion in  a  300  cc.  flask.  After  standing  ten  minutes  until  the  coarser 
sediment  had  fallen  to  the  bottom,  40  cc.  were  removed  and  placed  in 
two  sterile  centrifuge  tubes  and  centrifuged  for  five  minutes.  The 
supernatant  liquid  was  drawn  off  and  put  into  two  other  centrifuge 
tubes  and  centrifuged  for  thirty  minutes  at  2000  revolutions  per 
minute.  The  supernatant  liquid  was  drawn  off  and  discarded.  The 
last  5  cc.  of  the  liquor  and  sediment  were  thoroly  mixed  and  in- 
jected subcutaneously  into  guinea  pigs,  in  graded  doses,  giving  one 
il/2  cc.,  another  i  cc.,  and  for  the  first  test  a  third  received  y2  cc. 


RESULTS  OF       ^e  results  considering  the  length    of  time  that 
THE  TEST  tubercle  bacilli  live  in  the    soil    are    recorded    in 

Table  20.  The  guinea  pigs  inoculated  from 
samples  taken  on  the  day  of  exposure,  and  on  the  7th,  i6th  and 
34th  day  after  exposure  showed  in  each  case,  when  killed  and  ex- 
amined, severe  generalized  tuberculosis.  Microscopical  and  cul- 
tural tests  showed  the  germs  from  the  diseased  tissues  to  be  char- 
acteristic of  active,  living  tubercle  bacilli.  Tests  after  this  time  indi- 
cated a  weakening  in  virulence,  but  slight  tuberculosis  was  produced 
in  the  test  animals  from  material  taken  on  the  21  3th  day  of  expos- 
ure. Microscopic,  cultural,  and  guinea-pig  tests  from  the  diseased  tis- 
sues of  the  original  guinea  pig  showed  the  tubercle  bacilli  in  the  soil 
sample  to  be  active  and  virulent.  Five  tests  were  made  after  this 
date.  In  every  case  the  guinea  pigs,  when  killed  and  examined, 
were  found  to  be  healthy.  The  two  testings  after  the  last  one 
in  which  tubercle  bacilli  were  found,  viz.,  the  ones  made  on  the 


336 


BULLETIN  No.  161 


[November, 


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FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


337 


TABL.E  20—  Continued. 

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338  BULLETIN  No.  161  [November, 

2301!],  and  on  the  261  st  days  of  the  exposure,  produced  in  the 
third  and  fourth  weeks  an  enlargement  in  the  right  superior  in- 
guinals  of  each  guinea  pig  inoculated,  which  then  became  normal 
again.  Tho  it  cannot  be  surely  known,  it  may  be  that  the  number 
of  live  tubercle  bacilli  was  so  small  as  not  to  cause  the  disease,  but 
of  sufficient  number  to  produce  an  enlargement  of  the  glands. 
Three  tests  made  later  than  these  two  showed  no  such  enlargement 
of  these  glands. 

BOVINE  TUBERCULOSIS  IN  A  DEAD  ANIMAL, 
crtnorc  r»e          Besides  the  exposure  of  the  pure  culture  of  bovine 

bUUKL/t    \Jr  *    _  r 

MATERIAL  tubercle  bacilli  in  garden  soil,  a  guinea  pig  that 

had  died  of  bovine  tuberculosis  was  exposed  in 
this  same  soil.  This  guinea  pig  was  extensively  tuberculous.  It 
was  placed  in  a  flower  pot  and  covered  with  a  screen  of  wire  hav- 
ing a  mesh  fine  enough  to  exclude  earthworms.  The  screen  was 
pressed  down  close  over  the  guinea  pig,  which  allowed  the  fine  gar- 
den earth  to  come  into  immediate  contract  with  the  dead  body. 
The  abdominal  and  thoracic  cavities  had  previously  been  opened 
in  making  an  examination  of  the  guinea  pig  just  before  placing  it 
in  the  garden  soil. 

METHOD  OF  ^  ^e  time  of  the  first  test,  made  the  7ist  day  of 
TESTING  the  exposure,  the  tissues  of  the  guinea  pig  were 

so  decayed  that  the  flesh  and  the  skin  were  easily 
torn.  A  part  of  the  tuberculous  lung  and  of  the  right  superior  in- 
guinal lymphatic  was  removed  for  the  test.  At  the  next  test,  on 
the  99th  day,  most  of  the  soft  tissues  had  been  carried  away  by 
small  ants.  On  the  i33d  day  only  the  bones,  hair  and  some  ten- 
dons remained.  A  few  pieces  of  bones  and  a  bunch  of  hair  were 
obtained  for  this  test. 

The  results  of  these  tests  are  given  in  Table  21. 
RESULTS  Live,  active  tubercle  bacilli  were  found  on  the 

7 ist  day;  after  this  date  no  tuberculosis  was  pro- 
duced in  any  of  the  guinea  pigs  inoculated.  No  doubt  if  the  small 
ants  had  not  molested  the  soft  tissues,  tuberculosis  would  have 
been  produced  in  the  test  animals  at  later  dates.  While  no  final 
conclusion  can  be  drawn,  it  is  evident  that  these  germs  live  a  suffici- 
ent time  in  dead  tuberculosis  animals  to  be  dangerous  to  stock. 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


339 


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340  BULLETIN  No.  161  [November, 

DURATION  OF  LIFE  OF  TUBERCLE  BACILLI  IN  WATER 


PURPOSE  AND  ^ne  PurPose  °f  these  tests  was  primarily  to  deter- 
PLAN  OF  TESTS  mine  how  long  bovine  tubercle  bacilli  will  live  in 
a  watering  tank  such  as  is  found  on  the  average 
stock  farm  in  Illinois.  In  connection  with  this  work  it  was  desired 
to  test  the  length  of  time  human  tubercle  bacilli  will  live  in  drink- 
ing water.  With  this  in  view  experiments  were  planned  and  car- 
ried out  with  tubercle  bacilli  from  the  following  sources  : 

1.  Pure  cultures,  Series  i. 

2.  Pure  cultures,  Series  2. 

3.  The  diseased  tissues  of  a  tuberculous  guinea  pig. 

4.  Tuberculous  sputum. 

Series  i.    Pure  Cultures 

CULTURES  The  bovine  culture  was  the  same  as  that  used  in 

the  cow  manure  which  was  exposed  to  weather 
conditions.  The  human  culture  was  taken  from  the  tuberculous 
glands  of  the  neck  of  a  patient  from  the  Burnham  hospital  and  iso- 
lated in  this  laboratory.  The  tissues  were  received  May  25,  1910.  A 
pure  culture  was  obtained  by  inoculating  a  guinea  pig  and  cultur- 
ing  the  diseased  tissues  of  the  guinea  pig.  This  culture,  which  is 
typical  for  the  human  type,  has  since  been  kepi  upon  glycerine  agar. 

SAMPLES  OF       The  sample   °f  water  used  to  make  the   emulsion 
WATER  for  exposing  the   bovine  tubercle  bacilli    was  ob- 

tained from  the  large  watering  tank  used  to  water 
the  dairy  cattle  of  this  station.  This  tank  is  2^2x4x10  feet,  and 
is  supplied  with  running  water.  A  considerable  amount  of  spiro- 
gyra  and  various  kinds  of  smaller  green  algae  was  floating  in  the 
tank.  Diatoms  were  also  abundant.  A  green  scum  covered  a 
large  portion  of  the  surface  of  the  water.  The  sample  was  ob- 
tained in  a  500  cc.,  sterile,  cotton-stoppered  flask.  The  water  in 
the  tank  was  slightly  agitated  with  a  stick  about  as  much  as  it 
was  thought  the  cattle  Wtf'n*  stir  the  water  by  drinking.  The 
mouth  of  the  flask*  was  pi1  iQ'ed  under  about  six  inches  and  then 
allowed  to  fill.  The  sampl  -vas  at  once  taken  to  the  laboratory 
and  the  emulsion  prepared  aat  same  afternoon.  The  spirogyra 
died  soon  after  the  emulsion  was  made,  but  the  diatoms  and  the 
smaller  green  algae,  as  well  as  numerous  bacteria,  were  present  at 
the  conclusion  of  the  experiment  December  5,  1911,  586  days  after 
the  experiment  started.  The  sample  of  water  used  to  make  the  emul- 
sion with  the  pure  culture  of  human  tubercle  bacilli  was  taken  from 
the  tap  water  of  the  bacteriological  laboratory.  There  were  at  the 


19*-'] 


FATE  OF  TUHERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


341 


time  some  algae  and  bacteria  present  in  this  water,  a  part  of  which 
were  still  living  at  the  last  test  made. 


PREPARATION  emulsions    both  of  the    bovine  and  of    the 

OF  EMULSIONS  human  cultures  of  tubercle  bacilli  were  prepared 
in  the  same  way.  About  two  milligrams  of  a  four 
weeks'  culture  grown  upon  glycerine  agar  were  emulsified  in  the 
manner  described  under  ''Cultures  and  Emulsions,"  page  315.  The 
emulsion  of  bovine  tubercle  bacilli  was  diluted  to  350  cc.  with 
the  tank  water  obtained  for  this  purpose;  250  cc.  were  used  to 
expose  in  the  running  water,  and  100  cc.  to  expose  in  standing 
water.  In  a  similar  way  a  250  cc.  emulsion  of  human  tubercle 
bacilli  was  made. 


EXPOSURE   OF 
SAMPLE 


For  exposing  the  emulsions  in  running  water  two 
6-inch  flower  pots  were  obtained.  The  small 
opening  in  the  bottom  was  stopped  with  a  cork 
and  this  was  sealed  over  with  boiler  paint.  A  test  of  the  two 
flower  pots  thus  prepared  showed  that  they  held  water.  After 
immersing  them  for  twenty-four  hours  to  within  two  inches  of 
the  top,  no  water  rose  on  the  inside,  tho  it  became  very  moist.  The 
two  emulsions  were  now  poured  into  these  two  flower  pots  and 


FIG.  1.    THE  VESSELS  AND  THE  Poor,  OF  RUNNING  WATER  IN  WHICH  THE 
TUBERCLE  BACII^I  WERE  EXPOSED. 

The  intake  is  at  A;  the  outlet  at  B.  The  submerged  vase  C  has  been  placed 
upon  the  shelf  with  other  vessels  so  it  can  be  seen.  The  flower  pot  D, 
which  contained  the  tuberculous  guinea  pig,  is  placed  upon  a  brick  to 
bring  it  into  view.  The  two  unglazed  cylinders  and  one  of  the  flower 
pots  show  the  effect  of  freezing. 


342   - 


BULLETIN  No.  161 


[November, 


each  flower  pot  placed  in  a  gallon  glass  jar  containing  water  and 
immersed  about  three  inches,  so  that  the  level  of  the  emulsion  of 
tubercle  bacilli  on  the  inside  was  the  same  as  the  level  of  the  water 
on  the  outside.  The  water  in  the  glass  jars  around  the  outside  of 
the  flower  pots  was  kept  continually  running.  While  this  procedure 
does  not  give  the  same  condition  as  water  running  directly  into  the 
emulsion  of  bacteria,  it  allows  to  some  extent  the  circulation  of  the 
water  inside  the  porous  flower  pot  with  running  water  on  the  out- 
side of  these  vessels.  Another  part  of  the  emulsion  of  bovine  tu- 
bercle bacilli  was  kept  in  a  cotton-stoppered  glass  bottle.  This 
bottle  was  partly  immersed  in  one  of  the  glass  jars  of  running 
water.  The  water  could  in  no  way  circulate  in  this  bottle,  but  it 
was  kept  at  approximately  the  same  temperature  as  the  emulsions  in 
the  flower  pots. 

The  samples  were  kept  in  the  laboratory  until  April  15,  1911, 
250  days  after  the  emulsions  were  first  made.  It  was  then  found 
neither  convenient  nor  desirable  to  keep  them  there  longer.  A 
pool  was  prepared  in  the  courtyard  of  the  Agricultural  Building, 
to  which  they  were  transferred.  The  pool  was  made  by  sinking  a 
large  tile  three  feet  in  diameter  and  filling  in  the  bottom  with 
concrete.  Constantly  running  water  was  maintained  to  a  depth  of 
twenty  inches  the  year  round.  It  did  not  freeze  any  time  during 
the  winter  of  1911-12.  A  shelf  of  slate  was  placed  four  inches 
under  the  surface  of  the  water  on  which  the  vessels  containing  the 
tubercle  bacilli  were  set.  .The  pool  was  screened  against  flies  and 
other  insects.  (See  Figs,  i  and  2.) 


FIG.  2.    THE  POOL  SCREENED  AGAINST  FLIES. 


i<)i2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  343 

-reo-riMf  TUP      Nineteen  tests  from  these  samples  were  made  for 

TESTING  Tnt  ,A  . 

SAMPLES  the    presence  and    for    the  virulence    of  tubercle 

bacilli.  These  tests  were  made  on  the  9th,  23d, 
and  44th  days,  and  thereafter  about  once  a  month  until  the  last 
test,  which  was  made  December  5,  1911,  586  days  from  the  begin- 
ning. The  sample  of  the  bovine  tubercle  bacilli  kppt  in  the  cotton- 
stoppered  bottle  was  tested  for  only  202  days,  since  the  sample  was 
exhausted  at  this  time.  This  sample  was  not  transferred  to  the 
pool. 

Each  of  the  three  samples  was  tested  upon  the  same  days  and 
in  the  same  manner.  The  water  in  the  two  flower  pots  was  thoroly 
agitated  by  giving  it  a  circular  motion.  With  a  sterile  glass  rod 
the  sides  and  bottom  of  the  flower  pot  were  scraped  so  as  to  loosen 
adhering  sediment  that  might  contain  tubercle  bacilli.  The  water 
in  the  bottle  containing  the  bovine  tubercle  bacilli  was  thoroly 
shaken  before  the  test  sample  was  taken.  It  was  not  convenient 
nor  thought  necessary  to  rub  the  inside  of  this  bottle,  as  was  done 
in  the  case  of  the  flower  pots.  With  a  sterile  Pasteur  bulb  pipette 
from  each  of  the  three  containers  approximately  5  cc.  of  this  in- 
fected water  were  removed  and  placed  in  a  sterile  centrifuge  tube 
and  centrifuged  for  thirty  minutes  at  a  high  speed.  Four  cc.  of 
the  supernatant  liquid  were  removed  and  discarded.  One  or  two 
drops  of  the  sediment  were  placed  upon  a  glass  slide  and  a  micro- 
scopic preparation  made  and  stained  for  tubercle  bacilli.  The 
remaining  sediment  and  liquid  in  the  centrifuge  tubes,  about  i  cc. 
in  quantity,  was  thoroly  mixed  and  injected  subcutaneously  into  a 
guinea  pig.  The  same  precautions  were  taken  with  these  tests  as 
in  the  case  of  the  tuberculous  manure  to  guard  against  mistaking 
tubercles  produced  by  dead  tubercle  bacilli  for  those  produced  by 
living,  virulent  ones.  Microscopic  preparations  and  cultures  were 
always  prepared  to  test  the  diseased  tissues  of  the  infected  guinea 
pig  for  tubercle  bacilli.  In  a  part  of  the  cases,  however,  the  tuber- 
culosis was  so  extensive  that  it  was  not  thought  necessary  to  make 
inoculations  of  diseased  tissue  into  another  guinea  pig. 


RESULTS  OF  ^e  resu^ts  °f  the  tests  to  determine  the  length  of 
THE  TESTS  time  tubercle  bacilli  live  in  water  are  recorded  in 
Tables  22,  23  and  24.  With  the  first  four  tests 
of  each  of  the  three  samples  containing  tubercle  bacilli  the  guinea 
pigs  became  extensively  and  severely  tuberculous,  as  will  be  shown 
by  an  examination  of  the  three  tables.  Further  tests  of  the  dis- 
eased tissue  from  all  these  guinea  pigs  showed  the  tubercle  bacilli 
to  be  living-  and  virulent.  The  fifth  test,  made  after  126  days, 
showed  at  least  an  apparent  weakening  of  the  virulence  of  these 
germs. 


344 


BULLETIN  No.  161 


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i<)i2\  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  349 

The  tuberculosis  produced  in  the  two  guinea  pigs  inoculated  at 
the  fifth  testing  from  the  sample  of  standing  water  contained  in 
the  cotton-stoppered  bottle,  was  neither  extensive  nor  severe. 
Those  from  the  running  water  had  a  higher  virulence  than  the 
tubercle  bacilli  in  the  standing  water,  but  the  tuberculosis  produced 
was  not  so  severe  as  that  from  the  former  samples.  It  should  be 
noted  that  the  time  of  killing  these  guinea  pigs  was  much  earlier, 
and  it  may  be  for  this  reason  alone  that  the  virulence  appeared 
to  be  weakening.  This  fact  was  noted  at  the  time  of  killing  the 
four  guinea  pigs  inoculated  with  the  bovine  tubercle-bacilli-in- 
fected waters,  and  so  the  two  guinea  pigs  inoculated  with  water 
infected  with  the  tubercle  bacilli  of  the  human  type  were  not  killed 
at  this  time.  Thirteen  days  later  one  of  these  t\vo  guinea  pigs 
died  with  generalized  tuberculosis.  Unfortunately  the  death  of 
this  guinea  pig  was  not  noted  until  a  short  time  afterwards,  and 
it  could  not  be  determined  whether  tuberculosis  was  the  only  cause 
of  death.  The  other  of  the  two  guinea  pigs  inoculated  with  the 
sample  of  water  containing  the  human  type  was  killed  72  days 
later,  and  the  examination  showed  severe  generalized  tuberculosis. 

Later,  on  the  I47th  day,  we  find  severe  generalized  tuberculosis 
produced  from  each  of  the  three  samples.  After  this  date  only 
two  more  tests  were  made  from  the  sample  in  the  cotton-stoppered 
bottle,  one  on  the  iSoth  and  the  last  one  on  the  2O2d  day  of  ex- 
posure. These  two  tests  show  the  lessening  of  the  virulence  of 
the  germs :  for  from  the  former  no  tubercles  could  be  noted  24 
days  later,  when  the, two  inoculated  guinea  pigs  died  of  acute 
infection;  from  the  latter,  one  guinea  pig  died  too  early  to  make 
the  test;  the  other,  killed  78  days  after  inoculation,  showed  only 
a  doubtful  sign  Of  tuberculosis.  However,  an  inoculation  of  an 
emulsion  of  the  tissues  from  the  point  of  inoculation  and  the  right 
superior  inguinal  glands  produced,  in  a  secondary  guinea  pig,  gen- 
eralized tuberculosis.  From  the  diseased  tissues  of  this  guinea  pig 
an  active  culture  was  obtained.  Thus  no  end  point  was  reached 
from  the  sample  of  standing  water  with  the  bovine  tubercle  bacilli 
in  the  cotton-stoppered  bottle.  The  other  two  samples,  kept  in 
porous  flower  pots  which  were  transferred  to  the  pool  in  the  court- 
yard, remained  alive  for  441  days,  but  were  dead  on  the  47Oth  day. 

From  the  emulsion  of  the  bovine  type  severe  tuberculosis  was 
produced  in  the  guinea  pigs  even  in  the  last  test  in  which  these 
germs  were  found  alive.  From  the  emulsion  of  the  human  type 
only  localized  tuberculosis  was  produced  at  the  last  test.  The  sec- 
ondary guinea  pig  inoculated  from  the  diseased  tissues  of  this  last 
test  animal  showed  severe  generalized  tuberculosis,  and  cultures 
from  the  diseased  tissues  of  both  the  original  and  the  secondary 
test  animal  showed  these  germs  to  be  active.  No  test  made  later 
than  this  showed  any  indication  of  live  tubercle  bacilli.  Five  such 
negative  tests  were  made. 


350  BULLETIN  No.  161  [November, 

Scries  2.     Pure  Cultures 

Before  the  close  of  the  first  series  it  was  recognized  that  these 
experiments  were  of  such  importance  that  a  repetition  of  the  same 
was  advisable. 


PREPARATION     ^ie    cultures    used  and  the    preparation  of    the 
OF  EMULSIONS    emulsions  were  similar  to  that  in  Series  I. 

Emulsion  of  the  Human  type.  —  The  sample  of 
water  used  for  the  emulsion  of  the  human  type  was  obtained  from 
the  pool  June  15,  1911.  With  the  sample  was  obtained  also  a 
considerable  amount  of  floating  green  and  yellow  sediment  scraped 
from  the  inside  of  the  large  tiling  enclosing  this  pool  of  water.  A 
microscopic  examination  showed  very  abundant  algae  —  anabena, 
diatoms,  desmids,  scenedesmus,  and  confervoideae;  also  several 
kinds  of  bacteria.  Among  the  animalcule  there  were  vorticellae, 
amoeba,  a  few  paramoecium,  water-eels  and  rotifers. 

There  were  thoroly  shaken  450  cc.  of  this  algal  water  to  which 
was  added  an  emulsion  of  approximately  5  ing.  of  human  tubercle 
bacilli.  This  sample  was  divided  into  two  equal  portions:  one 
portion  was  placed  in  an  unglazed  earthen,  cylindrical  jar  eight 
inches  tall  and  three  inches  in  diameter;  the  other  portion  was 
placed  in  a  small,  unglazed  vase  which  was  stopped  tightly  with 
a  one-hole  rubber  stopper  in  which  was  inserted  a  small  glass  tube 
that  reached  above  the  surface  of  the  water  and  allowed  the  escape 
of  any  gases  that  might  accumulate  in  the  vase.  This  vase  was 
placed  on  the  bottom  of  the  courtyard  pool  twenty  inches  below 
the  surface  of  the  water.  The  other  sample  in  the  8-inch  jar  was 
placed  on  a  slate  shelf  four  inches  below  the  surface  of  the  water. 
This  jar  was  open  to  the  sunlight  but  the  direct  sun  could  not 
reach  the  surface  of  the  emulsion  inside.  During  the  winter  the 
part  of  this  jar  projecting  above  the  surface  of  the  water;  was 
crumbled  by  freezing.  (See  Fig.  i.) 

Emulsion  of  the  Bovine  type.  —  The  emulsion  of  the  bovine 
type  was  made  in  a  way  similar  to  that  of  the  human  type.  Five 
milligrams  of  these  organisms  from  a  young  culture  on  glycerine 
egg  were  removed  and  rubbed  up  thoroly  in  a  sample  of  225  cc. 
of  water  obtained  from  the  drinking,  trough  at  the  dairy  cattle 
barns,  and  thoroly  mixed.  A  microscopical  examination  showed 
the  following:  (a)  vegetation  —  spirogyra,  oscillaria  (large  and 
small  species,  the  small  species  being  very  abundant),  a  few  dia- 
toms, abundant  protococcus,  desmids,  scenedesmus  and  anabena  ; 
(b)  animalcule  —  amoeba,  paramoecia,  stylontia  and  vinegar  eels. 

This  sample  was  placed  along  with  the  emulsion  of  the  human 
type  in  a  similar  cylindrical  jar  on  the  slate  shelf  below  the  sur- 
face of  the  water  in  the  pool. 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


JS1 


TESTS 


>c        The  results  of   the  tests  of  the  bovine  and  the 

r  1-1  ill- 

human  types  exposed  in  the  open,  unglazed,  cylin- 
drical jars  are  given  in  Tables  25  and  26.  The 
last  test  in  each,  made  259  days  since  first  exposed,  showed  only 
local  tuberculosis  in  each  of  the  -four  guinea  pigs  inoculated.  Tho 
there  was  not  time  to  determine  by  cultures  and  by  secondary  in- 
oculations whether  these  organisms  were  living,  it  is  likely  that 
they  were.  The  end  point  in  this  series  was  not  reached.  The 
tuberculosis  produced  in  the  test  animals  from  all  other  samples, 
except  those  dying  with  acute  infection,  was  severe  and  general- 
ized. A  typical  autopsy  is  shown  in  the  case  of  Guinea  Pig  1124 
(Fig.  3),  which  was  inoculated  with  tubercle  bacilli  of  the  human 
type  after  exposure  for  212  days  in  running  water. 

The  results  of  the  tests  from  the  other  part  of  the  emulsion  of 
human  tubercle  bacilli  placed  in  the  submerged  vase  are  reported 
in  Table  27.  The  last  test,  made  on  the  25gth  day,  gave  gener- 
alized tuberculosis  in  the  two  guinea  pigs  inoculated. 


N» 


FIG.  3.    GENERALIZED  TUBERCULOSIS  IN  GUINEA  PIG  1124  INOCULATED  WITH  HUMAN 
TUBERCLE  BACILLI  AFTER  BEING  EXPOSED  IN  RUNNING  WATER  FOR  212  DAYS. 


352 


BULLETIN  No.  161 


[November, 


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FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


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FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


355 


The  Diseased  Tissues  of  a  Tuberculous  Guinea  Pig 

In  order  to  test  the  length  of  time^that  tubercle  bacilli  will 
live  when  exposed  in  running  water,  Guinea  Pig  918  was  selected. 
This  guinea  pig,  inoculated  with  tubercle  bacilli  of  the  human  type, 
was  killed  47  days  after  inoculation  and  found  to  be  severely 
tuberculous.  The  autopsy  showed  the  liver  and  the  spleen  to  be 
much  enlarged  and  thickly  set  with  yellow  masses  of  tubercles; 
the  lungs  and  lymphatics  were  also  extensively  infected.  Micro- 
scopical and  cultural  tests  showed  the  tubercle  bacilli  to  be  char- 
acteristic and  active. 


MANNEROF 
EXPOSURE 


The  dead  tuberculous  guinea  pig  was  placed  in  a 
flower    pot    having  the    opening   in   the  bottom 
closed.  A  half  brick  was  placed  upon  the  guinea 
pig  to  keep  it  it  from  floating  up  when  placed  in  the  running  water 
of  the  pool.     The  flower  pot  was  set  on  the  bottom  of  the  pool, 
which  was  twenty  inches  deep. 

RESULTS  ^^e  results  are  given  in  Table  28.     All  the  tests 

OF  TESTS  made  from  the    decaying  and  putrefying    tissues 

of  this  guinea  pig  with  one  exception  produced 


FIG.  4.    GENERALIZED  TUBERCULOSIS  IN  GUINEA  PIG  1126  INOCULATED  WITH  TUBERCU- 
LOUS SPUTUM  AFTER  BEING  EXPOSED  IN  RUNNING  WATER  FOR  187  DAYS. 


356 


BULLETIN  No.  161 


[November. 


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FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


357 


tuberculosis,  save  those  that  died  too  early  with  acute  infection. 
The  two  guinea  pigs  used  in  the  third  test,  made  99  days  af- 
ter exposure,  remained  healthy.  Probably  only  non-tuberculous 
tissue  was  obtained  for  this  test.  Later  tests  taken  from  broken- 
down  tissues  showed  virulent  tubercle  bacilli.  The  tests  were 
positive  up  to  the  32ist  day;  after  this  date  the  test  animals 
all  died  with  acute  infection,  the  last  one,  however,  not  too 
early  to  have  shown  tubercle  bacilli.  On  the  229th  day  and 
later  no  structure  of  tissue  could  be  determined  except  the  hair 
and  bones,  and  the  bones  were  almost  as  fragile  as  garden  earth. 
Sediment  from  the  bottom  of  the  flower  pot  was  obtained  for  the 
samples. 

Tuberculous  Sputum 

To  determine  the  length  of  time  that  tubercle 
bacilli  from  tuberculous  sputum  will  live  in  run- 
ning water,  a  sample  was  obtained  from  an  ad- 
vanced case  of  tuberculosis.  A  stained  smear  of  this  sputum 
showed  numerous  bacilli.  A  very  small  sample  was  inoculated  into 
a  guinea  pig  the  first  day  of  exposure.  This  guinea  pig  died  in 
three  days  of  acute  infection. 

RESULTS  The  results  are  given  in  Table  29.     Tuberculosis 

has  been  produced  in  the  test  animals  up  to  the 
last  test,  232  days  since  exposure.  However,  only  local  tubercu- 
losis was  produced  at  this  last  test  and  there  has  not  been  sufficient 
time  to  determine  whether  the  organisms  are  alive  or  dead.  The 
test  made  just  previous  to  the  last  one,  187  days  after  exposing 
the  sputum  in,  water,  produced  severe  generalized  tuberculosis  in 
Guinea  Pig  No.  1126  (see  Fig.  4). 


SOURCE  OF 
SAMPLE 


TABLE  29.    TUBERCLE  BACILLI  IN  WATER 


Kind  and  source  of  the  organisms 

Not  killed 

Killed 

Human  tubercle  bacilli: 
a.     Pure  culture  in  flower  pot,  Series  1  

441  days 

470  days 

b.     Pure  culture  in  8-inch  cylinder.  Series  2  
c      Pure  culture  in  submerged  va*e  

259  days 
259  days 

d      In  tuberculous  guinea  pig  •  

321  days 

381  days 

g      JQ  sputum  

232  days 

Bovine  tubercle  bacilli: 

441  days 

470  days 

b.     Pure  culture  in  8-inch  cylinder,  Series  1  
c.     Pure  culture  in  cotton-stoooered  bottle  .. 

259  days 
202  davs 

358 


BULLETIN  No.  161 


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I9i-]  FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY  359 


A  summary  of  the  results  showing  the  length  of  time  that 
tubercle  bacilli  from  various  materials -live  in  water  is  given  in 
Table  29. 

Conclusions 

1.  Tubercle  bacilli  live  for  more  than  a  year   (441  days)   in 
running  water. 

2.  The  length  of  time  that  human  and  bovine  tubercle  bacilli 
live  in  water  is  practically  the  same. 

3.  These  organisms  live  in  sputum  exposed  in  water  for  more 
than  232  days. 

4.  They  live  in  the  tissues  of  a  dead  guinea  pig  exposed  in 
water  for  more  than  321  days  but  are  dead  in  381  days. 

5.  A  watering  trough  harboring  tubercle  bacilli  may  become  a 
dangerous  source  of  infection  to  cattle. 

6.  A  dead  tuberculous  animal  in  a  stream  on  your  neighbor's 
farm  may  be  a  means  of  infecting  your  stock. 

7.  The  better   disposition   of   dead  tuberculous   animals   is   to 
destroy  by  burning. 

8.  Tubercle  bacilli  in    drinking  water    is  one  of  the    possible 
sources  of  infection  for  man. 

9.  Infection  is  not  prevented  by  dilution,  since  clumps  contain- 
ing a  great  number  of  these  organisms  may  be  inclosed  in  mucoid 
material  which  prevents  their  separation  and  destruction. 

DURATION  OF  LIFE  OF  BOVINE  TUBERCLE  BACILLI  IN  BUTTER 

INTRODUCTORY  Considerable  attention  is  at  present  being  directed 
STATEMENT  to  the  presence  of  tubercle  bacilli  in  foods,  more 
especially  milk,  butter  and  cheese.  It  has  been 
determined  by  Sedgwick  and  Winslow  and  by  Park  that  typhoid 
bacilli  frozen  in  water  die  very  rapidly.  After  an  hour's  freezing 
30  to  60  percent  were  destroyed,  and  in  two  weeks  99  percent  were 
killed.  The  remaining  one  percent  lived  for  a  number  of  weeks. 
Tubercle  bacilli  in  butter  kept  at  a  temperature  below  freezing  are 
not  killed  in  this  way,  as  determined  by  Mohler,  Washburn  and 
Rogers  in  1909.  In  order  to  obtain  further  information  upon  this 
subject  the  following  experiments  were  planned. 

Butter  was  mixed  with  an  emulsion  of  a  pure  culture  of  bovine 
tubercle  bacilli  and  placad  in  small  vials  which  were  stored  in  the 
three  following  places: 


360  BULLETIN  No.  161  [November, 

1.  The  cold  storage  of  the  Monarch  Refrigerating  Company, 
Chicago,  111.,  at  10°  C.  below  freezing. 

2.  The  University  of  Illinois  Dairy  storeroom  at  4°  C.  (above 
freezing). 

3.  The  basement  of  a  dwelling  in  Urbana,  Illinois,  kept  at  an 
average  of  approximately  20°  C. 

PREPARATION  ^  pound  of  fresh  butter  was  obtained  from  the 
OF  SAMPLES  creamery  of  the  University  of  Illinois  directly 
from  the  moulding  board.  It  was  salted  as  usual 
for  the  market,  one  ounce  of  dry  salt  to  one  pound  of  butter. 
After  the  butter  is  mixed,  pressed  and  drained  it  has  a  salt  content 
of  two  to  three  percent.  It  was  not  chilled,  but  at  once  taken  to 
the  bacterioligocal  laboratory  and  mixed  with  the  emulsion  of 
tubercle  bacilli.  An  emulsion  of  3  mg.  of  bovine  tubercle  bacilli 
in  100  c.c.  of  0.8  percent  salt  solution  was  made,  and  the  butter 
melted  at  35°  C.  was  thoroly  shaken  with  this  emulsion.  From 
about  10  to  15  cc.  of  this  emulsion  were  put  into  small  sterile  glass 
vials  and  stopped  with  sterile  cork  stoppers.  Thirty  such  samples 
were  prepared,  ten  of  which  were  stored  in  each  of  the  three  places 
mentioned  above. 


TESTING  THE      ^e  samples  were  tested  when  prepared  and  at 
SAMPLES  varying  intervals  afterward  on  the  same  day  from 

each  of  the  places  stored.  They  were  brought 
to  the  laboratory,  melted  at  a  temperature  of  about  38°  C.,  and 
two  guinea  pigs  were  each  injected  subcutaneously  with  i  cc.  of 
the  melted  butter  from  each  sample.  The  samples  kept  at  Chicago 
at  10°  C.  below  freezing  were  always  in  excellent  condition;  those 
kept  in  the  basement  of  the  dwelling  became  very  rancid  and 
slightly  mouldy;  and  those  kept  in  the  University  Dairy  storage 
showed  slight  moulding  in  part  of  the  bottles. 

RESULTS  The  results  of  the  tests  are  given  in  Tables  31,  32 

33.  No  end  point  was  reached.  Generalized  tu- 
berculosis was  produced  in  the  test  animals  from  each  of  the  three 
samples  taken  on  the  274th  day.  It  was  noted  that  the  tuberculo- 
sis produced  by  the  samples  kept  at  the  lower  temperature  was 
more  severe.  This  was  probably  due  to  the  killing  out  of  other 
organisms  that  at  higher  temperatures  acted  antagonistically  to  the 
tubercle  bacilli. 


1912} 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  TriE  ANIMAL  BODY 


361 


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FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL  BODY 


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364  BULLETIN  No.  161  [November, 

GENERAL  DISCUSSION 

A  summary  of  the  results  of  the  experimental  work  is  given 
in  Table  34.  It  is  seen  from  this  table  that  tubercle  bacilli  in  pure 
culture,  spread  in  thin  layers  on  sterile  glazed  paper  slips  and  ex- 
posed to  the  direct  rays  of  the  sun,  are  killed  in  a  very  short  time 
(i  to  4  minutes).  In  this  respect  tubercle  bacilli  may  be  classed 
with  other  nonspore-bearing  organisms.  When  exposed  to  desic- 
cation, pure  cultures  of  these  germs  in  thin  layers  are  found  to  be 
dead  in  a  few  days.  In  sputum  and  other  foul  material  they  appear 
to  live  longer  than  the  other  nonspore-bearers.  They  are  known 
to  live  long  enough  to  be  blown  around  so  that  the  inhalation  of 
dried  sputum  dust  causes  tuberculosis  in  test  animals.  Just  how 
frequently  people  are  infected  by  breathing  dried  tuberculous  ma- 
terial has  been  discussed  (see  page  270).  That  sunlight  plays  an 
important  part  in  the  disinfection  of  this  dried  tuberculous  dust 
is  evident.  Also  that  our  homes,  factories,  and  places  of  business 
should  have  an  abundance  of  window  space,  located  so  as  to  admit 
the  light,  is  another  timely  lesson. 

Tubercle  bacilli  in  cow  manure  lived  73  days  when  a  pure  cul- 
ture mixed  in  a  sample  of  manure  was  exposed  to  weather  condi- 
tions in  a  pasture  field  in  the  shade,  and  as  long  as  49  days  when 
exposed  in  the  sunshine.  The  sample  from  a  tuberculous  cow  was 
dead  at  the  first  test,  made  13  days  after  exposure.  We  were  dis- 
appointed in  not  being  able  to  repeat  these  experiments.  It  would 
be  advisable  to  repeat  this  work,  especially  with  naturally  infected 
manure  from  several  tuberculous  cows  that  were  known  to  be 
expelling  tubercle  bacilli  per  rectum.  Keep  pigs  from  three  to  four 
months  old  in  a  pasture  with  such  tuberculous  cows,  and  allow  the 
pigs  to  feed  upon  the  cow  dung.  Feed  other  pigs  by  mixing  with 
their  feed  tuberculous  manure  which  had  remained  in  this  pasture 
for  varying  intervals  of  time.  An  examination  of  the  internal  or- 
gans of  these  shoats  would  give  valuable  information  both  as  to 
the  infectiveness  of  the  cow  manure  and  to  the  length  of  time  that 
tubercle  bacilli  remain  alive  in  such  manure.  This  would  indicate, 
much  more  completely  than  our  experiments,  the  length  of  time 
that  stock  should  be  kept  from  a  field  in  which  tuberculous  cattle 
had  been  pastured. 

When  tubercle  bacilli,  either  in  manure  or  in  dead  tuberculous 
animals,  become  mixed  in  the  soil,  the  danger  may  be  still  greater, 
depending  upon  the  opportunity  for  hogs  to  take  this  material 
along  with  their  food.  The  bacilli  live  longer  under  these  condi- 
tions, but  the  opportunity  of  being  taken  is  usually  less. 

The  danger  of  man  becoming  infected  with  tuberculosis  from 
drinking  water  has  been  discussed  (see  page  307).  Just  how  likely 


FATE  OF  TUBERCLE  BACILLI  OUTSIDE  THE  ANIMAL 


BODY 


365 


TABLE  34.  —  SUMMARY  ox  RESULTS 


Organisms  exposed  to  direct  sunlight 


Name  of  organism 


B.  subtilis,  spores 

B.  mesentericus  vulgatus,  spores. 

B.  subtilis,  vegetative  cells   

B.  mesenteric,  vegetative  cells... 

B.  prodigiosus 

B.  diphtheria 

B.  coli 

B.  ty phosus 

B.  violaceus  . . 


Not  killed 


180  min. 
Yz  min. 


6  min. 
2  min. 

1  min. 

2  min. 


Killed 


1  min. 

%  min. 
after  6  min: 
3  min. 

2  min. 

3  min. 


Tubercle  Bacilli  Exposed  to  Direct  Sunlight 


Name  of  culture 


Not  killed 


Killed 


B.  tuberculosis,  human. 
B.  tuberculosis,  bovine 
B.  tuberculosis,  avian.. 


1  min. 

1  min. 

2  min. 


2  min. 
2  min. 
4  min. 


Bovine  Tubercle  Bacilli  in  Cow  Manure 


Kind  and  source  of  material  exposed 


Not  killed 


Killed 


Bovine  tubercle  bacilli,  pure  culture: 

Exposed  in  cow  manure,  in  sunshine,  Series  1. 
Exposed  in  cow  manure,  in  sunshine,  Series  2. 
Exposed  in  cow  manure,  in  shade,  Series  1  . . . . 
Exposed  in  cow  manure,  in  shade,  Series  2 


49  days 
45  days 
49  days 
73  days 


81  days 

73  days 

81  days 

100  days 


Bovine  Tubercle  Bacilli  from  a  Naturally  Tuberculous  Cow 


Tuberculous  manure  from  this  cow,  in  sunshine. 
Tuberculous  manure  from  this  cow,  in  shade  .... 


13  days 
43  days 


Bovine  Tubercle  Bacilli  in  Garden  Soil 


Bovine  tubercle  bacilli,  pure  culture 213  days  230  days 

Bovine    tubercle    bacilli,    in    tissue    of    a    dead 

guinea  pig 77  days  91  days 

Tubercle  Bacilli  in  Water 

Human  tubercle  bacilli: 

Pure  culture,  in  flower  pot,  Series  1 441  days  470  days 

Pure  culture,  in  8-inch  cylinder 258  days 

Pure  culture,  in  an  unglazed  vase,  submerged.    259  days 

In  the  tissues  of  a  tuberculous  guinea  pig 321  days  381  days 

In  tuberculous  sputum 232  days 

Bovine  tubercle  bacilli: 

Pure  culture  in  flower  pot,  Series  1 441  days  470  days 

Pure  culture,  in  an  8-inch  cylinder \  259  days 

Pure  culture,  in  a  cotton-stoppered  bottle j  202  days 

Bovine  Tubercle  Bacilli  in  Market  Butter 
Temperature  stored  Not  killed  Killed 

10°  C.  below  zero  , .    . .    274  days 

4°  C.  above  zero 274  days 

20°  C.  above  zero 274  days 


366  BULLETIN  No.  161  [November. 

it  is  that  cattle  and  other  farm  animals  are  infected  with  tuberculo- 
sis from  the  presence  of  these  germs  in  water  is  not  surely  known. 
The  common  watering  tank  may  become  a  source  of  infection.  Here 
the  tubercle  bacilli  live  among  the  algae  and  in  the  decaying  organic 
matter  for  more  than  a  year.  Calmette  points  out  that  constant 
and  repeated  infections  are  the  most  dangerous.  Cattle  would  thus 
be  subjected  when  a  watering  trough  was  infected. 

Another  source  of  danger  to  man  is  in  the  use  of  phosphates 
made  by  grinding  up  dead  tuberculous  animals  (which  is  done 
rather  extensively  in  the  United  States),  this  fertilizer  often  being 
used  in  vegetable  gardening.  One  can  easily  conceive  how  a  small 
piece  of  tuberculous  tissue  containing  many  dozens  of  tuberculous 
germs  could  be  made  to  adhere  to  an  onion  or  a  radish,  especially 
in  a  slightly  bruised  place,  and  be  carried  directly  to  the  consumer. 
That  these  germs  would  remain  alive  and  virulent  during  such  a 
circuit  there  is  no  question. 

It  is  seen  that  tubercle  bacilli  in  butter  kept  at  10°  C.  below  zero 
retain  their  virulence  longer  than  when  kept  at  the  higher  temper- 
ature. This  temperature  of  — 10°  C.  apparently  has  no  injurious 
effect  on  these  germs,  while  the  antagonism  of  other  organisms 
is  largely  prevented.  Butter  can  be  kept  in  cold  storage  for 
months  in  an  excellent  condition,  but  this  in  no  way  lessens  the 
danger  from  tubercle  bacilli  that  were  originally  introduced  into 
the  butter.  All  such  dairy  products  should  be  tested  by  govern- 
ment officials  not  only  for  quality  but  also  for  the  presence  of  tu- 
bercle bacilli. 

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